MicroRNA cargo of extracellular vesicles as mediators of immune dysregulation in pediatric immune thrombocytopenia: from pathogenetic drivers to biomarkers

1 Introduction

Immune thrombocytopenia (ITP) is an acquired autoimmune disorder affecting children and adolescents aged between 1 and 18 years, which leads to the production of autoantibodies against platelets. This condition causes thrombocytopenia, with a platelet count below 100 × 10⁹/L and an increased risk of minor bleeding (Di Paola et al., 2022; Gotesman et al., 2024; Singh et al., 2021). Pediatric ITP is usually an acute, self-limiting condition, characterised by a higher rate of spontaneous remission, but around 20%–30% of patients progress to a chronic form requiring long-term management (Despotovic and Grimes, 2018; Lee, 2023; Thakur et al., 2024). In general, ITP is classified as newly diagnosed within 3 months of onset, persistent if it lasts between 3 and 12 months, and chronic if it lasts for more than 12 months (Provan et al., 2019). First-line therapy consists of a watch-and-wait approach in newly diagnosed ITP pediatric patients with mild or no bleeding (Despotovic and Grimes, 2018). When necessary, intravenous immunoglobulin (IVIG) and glucocorticosteroids, such as prednisone, methylprednisolone, or dexamethasone are administered. In cases of chronic ITP refractory to first-line therapies, combining first-line treatments or thrombopoietin receptor agonists (TPO-RAs), including romiplostim and eltrombopag, represent second-line treatment options (Russo et al., 2024). The therapeutic effect of TPO-RAs is based on the activation of the thrombopoietin (TPO) receptor, which promotes the proliferation and maturation of megakaryocytes and platelet production (Wang Y. et al., 2026). For patients who respond poorly to TPO-RAs, rituximab, an anti-CD20 monoclonal antibody, is commonly recommended as a therapeutic option, as it exerts its immunosuppressive activity through B-cell depletion (Russo et al., 2024; Bussel and Garcia, 2022). ITP patients exhibit profound alterations in both their inflammatory and immune profiles, with a prevalence of the pro-inflammatory M1 macrophage phenotype and a high ratio of pro-inflammatory T helper 1 cells/anti-inflammatory T helper 2 cells (Th1/Th2), resulting in impaired inflammatory profiles and immune responses. In addition, mesenchymal stromal cells (MSCs), multipotent non‐haematopoietic cells with anti‐inflammatory and immunomodulatory properties, are strongly defective in ITP (Di Paola et al., 2020; He et al., 2021) (Figure 1). MicroRNAs (miRNAs) are short non-coding RNA molecules (19–25 nt) that regulate gene expression by silencing specific target transcripts and widely recognized as key regulators of immune homeostasis (Zhao et al., 2022). Extracellular vesicles (EVs) are a population of heterogeneous, membrane-bound carriers containing complex cargo, such as proteins, lipids, and nucleic acids, including miRNAs, which can be released by a variety of cells under physiological or pathological conditions (Payandeh et al., 2024; Villar-Vesga et al., 2019). These mechanisms highlight the need for dedicated studies on EV-associated miRNA (EV-miRNAs). In this context, EVs derived from megakaryocytes, platelets, and stromal cells may contribute to immune regulation and platelet homeostasis, transferring functional miRNAs (D'Amico et al., 2025). Recent advances have highlighted two complementary aspects of ITP pathophysiology: the role of the miRNAs as post-transcriptional regulators of immunity, and the function of EVs as key mediators of intercellular communication. A recent review has exhaustively summarised the importance of miRNAs in ITP, placing particular focus on immune dysregulation, megakaryocytic insufficiency, the potential of biomarkers and functional evidence (Liu et al., 2026). However, how miRNAs and EVs interact remains an underexplored area in pediatric ITP. In this review, we explore the landscape of deregulated miRNAs in ITP, with particular attention to their role in immune dysregulation, and the contribution of EVs as carriers of pathogenic miRNAs. A deeper understanding of the EV-miRNA signature may represent a potential diagnostic and therapeutic tool in pediatric ITP.

2 Deregulated miRNAs in ITP: a molecular landscape

miRNAs function is to silence gene expression by binding to partially complementary sequences in the 3’ untranslated region of target transcripts, leading to mRNA degradation or inhibition of translation (Zhao et al., 2022; O'Brien et al., 2018). miRNA genes are first transcribed into a primary transcript, known as pri-miRNA, and then cleaved by a microprocessor complex consisting of DiGeorge syndrome critical region gene 8 (DGCR8) and Drosha into a ∼70-nt stem-loop precursor, called pre-miRNA, within the nucleus (Bofill-De Ros and Vang Ørom, 2024). Next, the pre-miRNA moves to the cytoplasm via the Exportin 5 (XPO5)/RanGTP complex, where it is processed by the RNase III endonuclease Dicer into a mature ∼21-nt miRNA duplex (Bofill-De Ros and Vang Ørom, 2024; Kim et al., 2016). Only one of the strands of the duplex is chosen to be the guide and loaded into Argonaute proteins (AGOs) to assemble the RNA-induced silencing complex (RISC), which orchestrates the whole gene silencing machinery (O'Brien et al., 2018; Bofill-De Ros and Vang Ørom, 2024). Emerging evidence suggests that miRNAs are indispensable for maintaining immune system balance, thus when this regulatory machinery fails, miRNAs drive the pathogenesis of various autoimmune diseases, including ITP (Artimovič et al., 2024; Gaál, 2024). Altered miRNA expression disrupts immune tolerance, referring to the ability of the immune system to distinguish between the self and the non-self, lymphocyte subtype differentiation and platelet production (Zhao et al., 2022). Table 1 summaries some miRNAs which, according to reports, are deregulated in pediatric ITP, highlighting the available evidence regarding their association with the pathogenesis of the disease and their functional contribution to it.

TABLE 1. An overview of the various deregulated miRNAs in pediatric ITP. The table summarizes some of the miRNAs found to be deregulated in pediatric ITP. The altered expression of these miRNAs, in turn, leads to deregulation of their respective targets and pathways. Their role in contributing to the pathogenesis of ITP is described.

2.1 miR-155: the central regulator of inflammation

Despite the well-documented evidence of miR-155 as an oncomiR in T-cell lymphomas and various solid tumors, it also acts as a key factor in immune and inflammatory dysfunction in ITP (Chang et al., 2020; Mahesh and Biswas, 2019; Qian et al., 2014). miR-155 is expressed in haematopoietic stem and progenitor cells (HSPCs) and in mature haematopoietic cells, which include monocytes, granulocytes, B cells and T cells, and acts as a key regulator of immune cell responses (Alivernini et al., 2017). miR-155 contributes to T cell activation and differentiation and to the regulation of the Treg/Th17 balance, thus influencing pro-inflammatory immune responses. It regulates B cell proliferation and activation, germinal center formation, and antibody production (Alivernini et al., 2017). Its expression is upregulated in peripheral blood mononuclear cells (PBMCs) of adult ITP patients and is inversely correlated with both platelet count and anti-inflammatory mediators, including cytokine signaling suppressor 1 (SOCS1) mRNA, interleukin (IL)-4, IL-10, and transforming growth factor β1 (TGF-β1). On the contrary, it is positively correlated with plasma levels of the pro-inflammatory cytokine IL-17A (Qian et al., 2014). Recent studies focusing specifically on pediatric ITP have also reported increased expression of miR-155 in PBMCs from pediatric patients with newly diagnosed ITP compared with healthy controls, with higher expression observed in patients with severe disease. In this pediatric cohort, miR-155 expression was positively associated with the pro-inflammatory T helper 17 (Th17)/anti-inflammatory regulatory T (Treg) ratio and negatively associated with Treg cells and platelet count, suggesting an association between miR-155 expression and an imbalance in T cell subsets in pediatric ITP (Wang Z. et al., 2026). In the context of ITP, the available evidence mainly concerns cell-associated miR-155 expression, while its potential association with EVs and EV-mediated transfer remains to be clarified. In other immune contexts, miR-155 has been shown to modulate Th17/Treg balance and Th17 cell function through targeting SOCS1, supporting a potential role of this pathway in inflammatory immune dysregulation (Alivernini et al., 2017). In other inflammatory contexts, miR-155 hyperactivates the JAK-STAT (JAnus Kinase-Signal Transducers and Activators of Transcription) signaling pathways by directly suppressing negative regulators such as SOCS1, thereby blocking the resolution of inflammatory cascades (Bidgood et al., 2024; Wang et al., 2018). Consequently, targeting this axis represents a promising therapeutic strategy. Consistent with these findings, it has been demonstrated that in PBMCs and macrophages from ITP patients, miR-155-5p was upregulated whereas SOCS1 was downregulated. In vitro gain- and loss-of-function experiments showed that inhibition of miR-155-5p or upregulation of SOCS1 promoted polarization of macrophages towards the M2 phenotype, as indicated by an increased M2/M1 ratio, through the SOCS1-dependent Programmed cell death protein 1 (PD1)/Programmed cell death ligand 1 (PDL1) pathway (Chang et al., 2020). Overall, clinical evidence in pediatric ITP supports an association between increased miR-155 expression, inflammatory T cell imbalance, and disease severity, whilst the mechanistic evidence regarding the miR-155/SOCS1 axis still derives largely from studies conducted in adults and in vitro. Further functional studies in pediatric ITP are needed to determine whether these mechanisms are conserved in pediatric patients and whether they have therapeutic relevance.

2.2 miR-146a: the inflammation brake

miR-146a serves as a critical negative regulator of inflammatory responses, limiting excessive signalling by the innate immune system and maintaining immune homeostasis. Its anti-inflammatory function is primarily mediated through the repression of key signaling molecules involved in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, thereby providing a negative-feedback mechanism that prevents protracted inflammatory responses (Gilyazova et al., 2023). miR-146a targets and represses two specific targets: TNF receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK1), two signal transducers upstream of NF-κB (Gilyazova et al., 2023). Following stimulation of cell surface receptors, like the interleukin-1 receptor (IL-1R) or members of the Toll-like receptor (TLR) family, downstream signaling is activated via the MyD88-dependent pathway, which recruits IRAK1 and TRAF6 (Pe et al., 2023). They trigger a cascade that leads to the activation and nuclear translocation of NF-κB, where it induces the transcription of various target genes, including pro-inflammatory chemokines IL-8, C-X-C motif chemokine ligand 1 (CXCL1), and Chemokine C-C motif ligand 5 (CCL5), as well as the primary transcript pri-miR-146a (Gilyazova et al., 2023; Rusca and Monticelli, 2011). A negative feedback loop is established, where the mature miR-146a suppresses IRAK1 and TRAF6 expression. The downregulation of these two targets attenuates the subsequent NF-κB-mediated cytokine production, thereby preventing inflammatory hyperactivation. In a non-pediatric ITP cohort, miR-146a expression was downregulated and positively correlated with Treg cell frequency and platelet count and negatively correlated with Th17 cell frequency (Liu et al., 2016). In pediatric patients with ITP reduced plasma miR-146a-5p levels were reported in both newly diagnosed and chronic ITP compared with healthy controls (El Zaiat et al., 2025). These findings concern circulating miR-146a-5p and do not, in themselves, establish its association with EVs. EVs derived from bone marrow MSCs (BMSCs) offer a method for delivering functional miR-146a-5p to target CD4⁺ T cells, helping to correct the Th17/Treg imbalance observed in ITP through the direct targeting and repression of endogenous IRAK1 (He et al., 2021). The restoration of the miR-146a-5p/IRAK1 pathway mediated by BMSC EVs thus provides a viable cell-free therapeutic approach for clinical intervention (Figure 2). In addition to this link to the Th17/Treg axis, miR-146a-5p may also contribute to the Th1/Th2 balance. In pregnant patients with ITP, reduced levels of miR-146a-5p have been associated with increased expression of Caspase recruitment domain family member 10 (CARD10), while miR-146a-5p derived from BMSC-EVs was shown to modulate the Th1/Th2 balance via the CARD10/NF-κB pathway. Consistent with this mechanism, it has been demonstrated in vitro that CARD10 suppresses Th2 polarization and enhances Th1 responses, potentially contributing to the pro-inflammatory environment associated with ITP (Liu et al., 2026; Rong et al., 2024). Therefore, while clinical studies support an association between reduced miR-146a-5p expression and ITP, including pediatric ITP, the proposed mechanisms involving IRAK1 and CARD10 remain largely supported by experimental evidence.

2.3 miR-142: a role in immune tolerance

miR-142 is expressed preferentially among haematopoietic cells, playing a key role in haematopoietic homeostasis (Huang et al., 2023). miR-142 contributes to the regulation of immune-cell activation, differentiation, and homeostasis (Olson and Derudder, 2023; Sharma, 2017). The expression of miR-142 is found to be deregulated in autoimmune diseases and hematological malignancies, such as leukemia and lymphoma (Huang et al., 2023). miR-142 knockout mice model displays a significant reduction in Treg cells in both thymus and secondary lymphoid organs, suggesting that it plays a key role in Treg cells development and stability (Wang et al., 2022). In Treg cells, miR-142-5p is the predominant isoform, whose target is cAMP-hydrolyzing enzyme phosphodiesterase-3b (PDE3B) mRNA (Anandagoda et al., 2019). PDE3B hydrolyzes its substrates cAMP, accelerating intracellular cAMP turnover. As the immunosuppressive function of Treg cells depends on a high cAMP intracellular concentration, PDE3B inhibition is required for the maintaining of these elevated levels (Anandagoda et al., 2019). Mechanistic studies have demonstrated that the specific deletion of miR-142 in Treg cells increased PDE3B expression, resulting in a decreased intracellular cAMP level and impaired suppressive function of Treg cells, whilst inhibition or genetic ablation of PDE3B restored the suppressive activity of Treg cells (Anandagoda et al., 2019). These observations suggest that the miR-142/PDE3B/cAMP axis participates in the maintenance of Treg cell function and peripheral immune tolerance. miR-142-3p is generally reported to be downregulated in adult ITP patients and shows a significant negative correlation with the frequency of Th17 cells (Liu et al., 2016). However, it was found to be upregulated in PBMCs of the ITP patient cohort examined by Qian C et al., probably due to differences in patient demographics, geographic origin, disease status, and the limited sample size of the study (Qian et al., 2018). Although the miR-142-5p/PDE3B/cAMP pathway provides a mechanistic basis for its role in Treg function, this mechanism has not been directly demonstrated in pediatric ITP. Overall, the resulting Th17/Treg imbalance promotes the persistent autoimmune response that causes increased ⁠platelet destruction.

3 EVs selective miRNA loading

For many years, EVs were considered cellular waste and received little attention. However, the development of standardized isolation and detection methods led to a rapid expansion of EV research (Aloi et al., 2024). EVs are a heterogeneous population of particles delimited by a lipid bilayer and released by diverse cell types, such as immunoregulatory cells, including MSCs, Treg cells, and dendritic cells, or tumor cells or tumor cells (Buzas, 2023; Kumar et al., 2024; Shah et al., 2025; Welsh et al., 2024). They are unable to replicate due to the absence of functional nuclear material and their production is often enhanced by specific signals, such as cell activation, oxidative stress, hypoxia, and other pathological conditions (Welsh et al., 2024; Żmigrodzka et al., 2016). EVs mediate intercellular communication and signaling by transporting a wide range of bioactive molecules, such as proteins, lipids, and nucleic acids to recipient cells and thereby modulating immune responses (Aloi et al., 2024; Kumar et al., 2024; Shah et al., 2025). Immunoregulatory cell-derived EVs can transfer the functional properties of their parent cells to recipient cells and influence the differentiation of immune cells, cytokine production and other aspects of the immune microenvironment, confirming their potential as cell-free immunomodulatory therapies in autoimmune diseases (Shah et al., 2025). EVs secreted under pathological conditions reflect the pathological states of their originating cells, and these profiles represent innovative, non-invasive biomarkers for early detection, prognosis and disease progression across a variety of conditions (Xu et al., 2022). In autoimmune diseases, these EVs act as messengers that can alter inflammatory responses (Chen et al., 2026; Duan et al., 2019).

EVs can be classified into small EVs (<200 nm in diameter) and large EVs (>200 nm) (Welsh et al., 2024; Lee et al., 2023). It is important to note this kind of classification may include mixed populations, as separation methods do not yield perfectly homogeneous vesicle subsets. They can be also classified by biogenesis into exosomes, ectosomes (also referred to as microvesicles or microparticles) and apoptotic bodies (Welsh et al., 2024). Exosomes (0.03–0.15 μm) originate from the endosomal system and are generated through the fusion of multivesicular bodies with the plasma membrane (Li et al., 2026). Ectosomes (0.1–1 μm) refer to EVs from the cell surface, generated by the outward budding of the plasma membrane during cell activation (Surman et al., 2017). Apoptotic bodies are larger vesicles (1–5 μm) released during programmed cell death and may contain nuclear and cellular fragments (Shi et al., 2025). Alternatively, EVs are also classified using their cellular origin, including platelet microparticles (PMPs), erythrocyte microparticles (RBC-MPs), endothelial cell microparticles (EMPs), and tumor cell microparticles (TMPs). Oncosomes are another type of EVs, representing large vesicles (1–10 μm) released by migrating tumor cells (Żmigrodzka et al., 2016) (Figure 3).

Unfortunately, these size- and biogenesis-based definitions should be applied cautiously, as current isolation methods do not allow complete separation of EV subtypes with distinct origins, and no universal markers for ectosome, exosome or other EV subtypes exist. EV classification remains a subject of ongoing debate, due to their different sizes, cellular origins, and biogenesis pathways and the limitations of current isolation and characterization techniques. The International Society for Extracellular Vesicles (ISEV) in the “Minimal information for studies of extracellular vesicles 2018 (MISEV2018)” statement officially recognises this heterogeneous population of vesicles under the collective term of extracellular vesicles (Welsh et al., 2024). For the purposes of this review, EVs will be used in a broad sense, focusing mainly on their miRNA content.

EVs are enriched with noncoding RNAs, including miRNAs, tRNA, rRNA, mitochondrial RNA, circular RNA (circRNA), and long noncoding RNA (lncRNA) (Liu and Halushka, 2025). Several databases, such as Vesiclepedia, ExoCarta, and EVpedia, have been developed to catalogue EV cargo. More recently, EVmiRNA2.0 was developed as an expanded platform to provide for the comprehensive analysis of EV miRNA expression profiles and their potential roles in pathological diseases (Xie et al., 2026). Several distinct molecular pathways govern the sorting of miRNAs into EVs, which can be broadly categorized into RNA-binding proteins (RBP)-dependent and RBP-independent mechanisms (Huang et al., 2025). RBPs, including AGO2 and heterogeneous nuclear ribonucleoproteins (hnRNPs), act as shuttles capable of targeting specific miRNAs to control the loading into EVs for extracellular transport (Liu and Halushka, 2025; Groot and Lee, 2020; Marocco et al., 2025; O'Grady et al., 2022). HnRNPs recognize and bind unique sequence motifs within the miRNAs, known as EXOmotifs, such as GGAG/UGCA, driving their sequestration into EVs (Huang et al., 2025; Villarroya-Beltri et al., 2013). Recent findings indicated that AGO2-miRNAs represent the majority of free human plasma miRNAs, suggesting that AGO2 might have an important role in stably binding and sorting miRNAs into EVs (Groot and Lee, 2020). By contrast, RBP-independent sorting mechanisms have been associated with lipid-based microdomains, such as cholesterol and sphingolipid-enriched membrane regions, which serve as platforms for the passive EV incorporation of miRNAs (Huang et al., 2025). The encapsulation of miRNAs within EVs contributes to their stability and prevents degradation by extracellular RNases (Yáñez-Mó et al., 2015). Upon reaching their destination, EV-miRNAs can then be taken up by recipient cells through various mechanisms, including endocytosis, phagocytosis, or direct fusion with the plasma membrane, depending on the cell type and the surrounding microenvironment (Guo et al., 2024). EV-miRNAs modulate essential processes such as proliferation, differentiation, apoptosis, and immune responses, contributing to both physiological homeostasis and the development of various diseases, due to their role in intercellular communication (Wu et al., 2026). Despite their therapeutic potential, the clinical application of EV-based therapies continues to be challenged by several difficulties, most notably the standardization of EV isolation and profiling, targeting efficiency, and the lack of a complete assessment of long-term safety (Shah et al., 2025).

3.1 Platelet microvesicles as miRNA carriers in ITP

Platelet-derived extracellular vesicles (PDEVs) constitute a heterogeneous population of vesicles released by platelets and their precursor cells, the megakaryocytes. Platelet-derived microvesicles, also known as platelet microparticles (PMPs), are generated predominantly through outward budding and detachment from the plasma membrane of activated platelets, whereas megakaryocytes can also release extracellular vesicles directly (Stone et al., 2022). PMPs represent a major population of circulating extracellular vesicles, characterized by a heterogeneous EV population ranging from 0.1–1 μm. Following platelet activation, they are produced and released from the cellular plasma membrane (Jolink et al., 2021). They retain platelet-derived membrane proteins and receptors and carry a diverse cargo of lipids, proteins, enzymes, and nucleic acids, including mRNAs and miRNAs, which can be transferred to recipient cells (Puhm et al., 2021). PMPs can influence the functions of recipient cells through these interactions, which are mediated by the substances they carry and are involved in intercellular communication, inflammatory responses and immune regulation (Puhm et al., 2021). In addition to their role in thrombosis and thrombopoiesis, PMPs may also be involved in immune regulation by interacting with macrophages and other immune cells. Recent experimental evidence indicates that platelet-derived EVs can modulate macrophage responses, with immunomodulatory effects that depend on the activation state of the parent platelets. Moreover, PMPs may contribute to the communication between platelets and immune cells and potentially influence adaptive immune responses through the transfer of bioactive molecules (Palviainen et al., 2024). However, these immunomodulatory effects have been mainly investigated in non-ITP contexts, and their specific contribution to the immune dysregulation of ITP remains to be established. Unlike the platelets, PMPs are able to easily enter the lymphatic system and bone marrow, where they can act as crucial epigenetic signals by transferring specific transcripts (Nelson et al., 2021). miR-1915-3p is highly enriched in PMPs and is transferred to HSPCs, where it directly represses the B member of the Rho GTPase family (RHOB), promoting megakaryocytic differentiation and platelet production. In vitro uptake of PMPs by HSPCs and the overexpression of miR-1915-3p have validated the miR-1915-3p/RHOB regulatory axis, while the administration of PMPs increased the number of megakaryocytes and platelets in vivo (Qu et al., 2020). It remains to be determined whether this PMP-mediated miR-1915-3p/RHOB pathway is altered in the pathological context of ITP. However, an altered PMP burden could potentially contribute to the dysregulation of thrombopoiesis and immune homeostasis within the bone marrow niche in ITP (Nelson et al., 2021). In many studies, miR-223-3p was described as one of the most abundant platelet-derived miRNAs (Charlon-Gay et al., 2026; Leng et al., 2022). miR-223 is a critical haematopoietic miRNA that serves as a master regulator of immune cell development, inflammation, predominantly enhancing megakaryocyte differentiation and maturation by inhibiting Myosin Heavy Chain 10 (MYH10) and LIM Domain Only 2 (LMO2) (Leng et al., 2022; Jiao et al., 2021). miR-223 also exerts its anti-inflammatory effects, maintaining homeostatic macrophage polarization. Experimental studies have shown that inhibition of miR-223 expression alters macrophage polarization, whereas restoration of its expression promotes anti-inflammatory M2 transition. Its targets are key regulatory metabolic pathways, including the transcription factor Hypoxia-inducible factor 1-alpha (HIF-1α), Signal transducer and activator of transcription 3 (STAT3), and the Forkhead box protein O1 (FOXO1)/Peroxisome proliferator-activated receptor gamma (PPARγ) signaling axes (Dang and Leelahavanichkul, 2020; Zhang et al., 2025). In ITP, an imbalance of M1/M2 ratio has been observed, with a prevalence of M1 macrophages, responsible for an increased release of pro-inflammatory cytokines, such as IL-6, TNF-α and Interferon-γ (IFN-γ), driving the underlying immune deficiency (Di Paola et al., 2020). Notably, altered miR-223-3p expression has also been reported in the plasma of pediatric patients with ITP, supporting its potential involvement in the disease context (Bay et al., 2014). However, despite its established roles in megakaryocyte and macrophage biology, the specific pathogenic contribution of this miRNA to ITP has not yet been established. Future research will need to isolate and specifically map the miRNA cargo contained within PMPs, as miRNAs are mostly measured as free circulating miRNAs in plasma and not specifically measured within PMPs. The evaluation of EV-miRNAs depends on pre-analytical conditions and EV isolation procedures. Residual platelets may co-isolate with EV-enriched plasma fractions and release additional platelet-derived miRNAs during sample processing, potentially confounding the quantification of EV-miRNAs. Indeed, platelet removal has been shown to improve the detection and reproducibility of EV-miRNA measurements. Therefore, standardized blood collection and processing, adequate control of platelet contamination, and EV characterization according to current MISEV recommendations are essential for the reliable evaluation of EV-miRNAs as biomarkers (Welsh et al., 2024; Bettin et al., 2022; Bracht et al., 2023). This targeted molecular understanding will pave the way for new diagnostic and therapeutic strategies for ITP.

3.2 Immune cell-derived EVs as miRNA Carriers in ITP

EVs produced by immune cells represent a crucial field of miRNA-mediated intercellular communication, which remains largely unexplored in ITP. Immune cell-derived EVs exhibit immunomodulatory effects ranging from interactions between immune cells to the balance of T helper subset and the polarization of macrophages (Wu et al., 2026). EVs could provide promising therapeutic platforms for the delivery of functional miRNAs, thanks to their high biocompatibility, low immunogenicity, intrinsic ability to cross biological barriers, and the possibility of being engineered for targeted delivery to specific cells or tissues (Liu et al., 2024; Luisotti et al., 2025). Among the various sources of EVs, those derived from MSCs have attracted particular interest due to their intrinsic regenerative and immunomodulatory properties and are currently being studied as cell-free therapeutic alternatives for both oncological and non-oncological diseases, including autoimmune diseases (Rajendran and Gangadaran, 2026; Zhou and Shan, 2026). MSCs are a heterogeneous cell type that can be isolated from a variety of various tissues, such as adult adipose tissue, peripheral blood, endometrial polyps, menstrual blood, bone marrow, neonatal placenta, and umbilical cord tissues, able to proliferate extensively and to differentiate into different cell types such as osteoblasts, adipocytes, and chondrocytes (Clark et al., 2014; Xu et al., 2024). MSCs exert their anti‐inflammatory and immunomodulatory functions by actively interacting with components of the innate immune system through secreting EVs, mainly showing anti-inflammatory effects (Luque-Campos et al., 2021). As previously stated, MSCs from ITP patients (ITP-MSCs) are functionally defective, displaying proliferation defects, increased apoptosis, and loss of immunoregulatory function, with an increased pro-inflammatory cytokine profile, which concur in ITP pathogenesis (Di Paola et al., 2020; He et al., 2021). Nowadays, miRNAs have emerged as pivotal regulators of MSCs proliferation, differentiation, and paracrine functions, previously described as impaired in ITP (Clark et al., 2014; Yang et al., 2025). miR-146a represents one of the most promising therapeutic candidates in ITP as it acts as a negative regulator of NF-κB signaling and is consistently downregulated in patients with ITP (He et al., 2021). The therapeutic potential of miR-146a-engineered EVs is supported by studies conducted on other inflammatory diseases, in which they effectively suppress the IRAK1/TRAF6/NF-κB signaling pathway and restore immune homeostasis, reinforcing their potential application in ITP (Yang et al., 2025; Shahryari et al., 2025) (Figure 2). Beyond miR-146a-5p, other MSC-derived miRNAs involved in ITP pathogenesis have been described. miR-98-5p represents an important pathogenic hub because it is increased in ITP-MSCs and promotes cell apoptosis by targeting insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), leading to the downregulation of its downstream target, insulin-like growth factor 2 (IGF-2) (Wang et al., 2020). Experimental modulation of miR-98-5p in ITP-MSCs confirmed its functional role: miR-98-5p overexpression increased MSC apoptosis, whereas its inhibition reduced apoptosis and restored IGF2BP1/IGF-2 (Liu et al., 2024). The subsequent downregulation of IGF-2 causes inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is involved in the process of MSC deficiency (Li et al., 2021). Additionally, miR-98-5p upregulates p53 by inhibiting β-transducin repeat-containing protein (β-TrCP)-dependent p53 ubiquitination. Importantly, the MSCs investigated in this study were derived from adult patients with ITP, and the relevance of the miR-98-5p/IGF2BP1/IGF-2 axis to pediatric ITP remains to be established. A further point of interest is the role of MSCs in promoting alternative regulatory functions, such as the induction of regulatory B cells, a subset of B cells (Chen et al., 2021). In this context, umbilical cord-derived MSCs (UC-MSCs) can promote regulatory B cells differentiation via miR-133-dependent EVs. Taken together, these findings suggest that MSCs should not be classified merely as dysfunctional targets within the ITP microenvironment, but rather as a source of therapeutic EVs capable of bringing about comprehensive immune remodulation (Figure 4).

4 AntagomiRs and miRNA mimics: new therapeutic frontiers in ITP

Recent advances in miRNA-targeted strategies have highlighted the role of the epigenetic profile in ITP, including DNA methylation, histone modifications, and non-coding RNA, and their potential as new therapeutic targets capable of influencing the course of the disease (Tan et al., 2023). miRNAs discussed in this section were selected based on the availability of experimental evidence supporting their therapeutic modulation using antagomiRs, miRNA mimics, or related miRNA-targeting approaches in ITP. AntagomiRs or miRNA mimics offer a promising targeted strategy for manipulating miRNA expression, aimed at restoring cellular homeostasis. AntagomiRs are small, chemically modified miRNA antagonists that act as direct or functional structural inhibitors, perfectly complementing the specific miRNA target (Guo et al., 2023; Lima et al., 2018). These function as artificial inhibitors of target miRNAs, by transiently repressing their expression. Following binding to the target miRNA, antagomiR causes steric hindrance, preventing the miRNA from entering the RISC complex (Tan et al., 2023). This, in turn, leads to the derepression of the target transcripts, restoring normal translation and proper cellular function. Various strategies have been developed for the delivery of antagomiRs, including the use of nanoparticles, cationic polymers, transfection or microinjection techniques, but EVs have attracted growing interest as a delivery platform due to their high biocompatibility, and their potential to facilitate the transfer of therapeutic cargo to recipient cells (Luisotti et al., 2025; Guo et al., 2023). However, the therapeutic application of EV-miRNA delivery still faces important challenges, including efficient and specific targeting of recipient cells, optimization of miRNA loading and release, and control of cargo stability. In addition, the ability of miRNAs to regulate multiple target transcripts raises concerns regarding potential off-target effects and inadvertent modulation of biological pathways (Luisotti et al., 2025; Seyhan, 2024). In contrast to antagomiRs, there are inhibitors that enable long-term miRNA inhibition. This is the case of miRNA sponge, a specially designed RNA transcript expressed by DNA vectors that competitively binds to the target miRNA (Avendaño-Portugal et al., 2025). Once transfected and stably expressed in cells, the miRNA sponge can exert prolonged or permanent suppression against a single or multiple target miRNAs. A promising therapeutic approach for ITP is miR-155 targeting, considering its overexpression and its central role in promoting Th1/Th17 response and inhibiting Treg cells in ITP (Chang et al., 2020; Qian et al., 2014; Yao et al., 2012). Thus, a specific inhibitor of this specific miRNA could represent a potential novel application to target its pathogenic role. This hypothesis is confirmed by the development of Cobomarsen (MRG-106), an oligonucleotide inhibitor already tested in human clinical trials for some onco-hematological diseases, such as lymphomas and leukemias (Seto et al., 2018). miR-641 upregulation in peripheral T cells of adult ITP patients is associated with Th17/Treg imbalance, because miRNA targets and inhibits the expression of Special AT-rich sequence-binding protein 1 (SATB1) and Stromal interaction molecule 1 (STIM1), two important regulators of T cell differentiation and immune homeostasis (Zhu et al., 2024). In experimental models, both miR-641 sponge in ITP primary T cells in vitro, or antagomiR-641 injected in vivo in a murine ITP model caused the restoration of the expression of these two transcripts and of the Th17/Treg balance, modulation of the cytokine response, and improvement in platelet count, highlighting its potential as a therapeutic target. miR-557 is overexpressed in the plasma of ITP pediatric patients and negatively regulates the proliferation and maturation of TPO-induced megakaryocytes. Wang Y et al. study has demonstrated that inhibition of miR-557 using an antagomiR activates the protein kinase B (Akt)/Extracellular signal-regulated kinases (ERK) signaling pathway, resulting in reduction of proportion of CD4⁺ T lymphocytes in PBMCs and increases the proportion of Treg cells in CD4⁺ T lymphocytes in peripheral blood of ITP model rats (Wang et al., 2021). In conclusion, the use of antagomiRs represents an innovative frontier compared to traditional treatments for ITP, because these molecules target the specific molecular pathogenic target without causing generalized immunosuppression.

In addition to antagomiRs, miRNA mimics, a double-stranded synthetic RNA, are widely used in the study of miRNA characteristics and their contribution to the pathogenesis of various diseases. Since its function is to restore the endogenous function of a target miRNA, a miRNA mimic is employed when the miRNA expression is downregulated (Luisotti et al., 2025; Seyhan, 2024). To better understand the effect of miR-199a-5p on immune cells, in a study conducted by Li J et al., 2021, CD4⁺ T lymphocytes isolated from the spleen tissues of healthy mice were transfected with the miR-199a-5p agomiR, a chemically modified miRNA mimic that enhances its stability (Li et al., 2021). From a functional perspective, its overexpression blocks Th17 cell differentiation by acting directly on STAT3. Consistent with this mechanism, miR-199a-5p-enriched EVs derived from MSCs in the adipose tissue (ADSCs) of healthy mice inhibit pro-inflammatory Th17 cell differentiation and improve platelet counts in experimental models of ITP, supporting the therapeutic potential of EV-mediated administration of miR-199a-5p. Similarly, the upregulation of miR-146a expression in ITP patients with agomiR significantly reduces the differentiation of Th17 cells and elevates the differentiation of Tregs, thereby restoring immune homeostasis (Liu et al., 2016). The expression of miR-99a in CD4⁺ T lymphocytes from adult patients with ITP is reduced, and its overexpression, achieved via transfection with a miRNA mimic, increased Treg levels in patients with ITP by repressing mTOR (Hua et al., 2019). Studies conducted on pediatric patients with ITP have revealed elevated plasma levels of the CXC inflammatory chemokine ligand-13 (CXCL13). CXCL13 concentration decreases following therapeutic treatments, such as dexamethasone. The introduction of a miR-125-5p mimic into CD4⁺ T lymphocytes is capable of acting directly on the expression of CXCL13 and suppress it, mimicking the therapeutic effect of corticosteroids, whilst the miR-125-5p inhibitor has the opposite effect (Li et al., 2015). Although challenges regarding delivery and stability remain, loading these synthetic molecules into EVs could represent a potential cell-free therapeutic strategy for restoring immune tolerance. However, the available evidence in ITP remains largely preclinical, with most studies based on in vitro experiments or animal models. Further studies are needed to establish the long-term safety, biodistribution, therapeutic efficacy, and optimal dosing of these approaches before their translation into clinical practice, particularly in pediatric ITP.

5 miRNAs in EVs as non-invasive biomarkers in pediatric ITP

In ITP, where platelets, T and B lymphocytes, monocytes, and megakaryocytes all contribute to pathogenesis, this specificity becomes particularly valuable. The diagnosis of pediatric ITP remains one of exclusion and there are no validated diagnostic, prognostic or predictive biomarkers of therapeutic response (Allegra et al., 2023). The EV-miRNAs profile reflects the specificity of the tissue of origin, retaining the biological markers of the cell of origin (Barger et al., 2016). However, an active sorting mechanism selects and enriches specific miRNAs in EVs, transforming these vesicles from simple passive biomarkers to active targeted mediators of intercellular communication (Mori et al., 2019). A relevant methodological aspect concerns the choice between the analysis of free miRNAs and EV-associated miRNAs as biomarkers. In order to predict whether a newly diagnosed pediatric patient will go into spontaneous remission or chronic condition, circulating miR-146a-5p, miR-106b-5p and miR-200c-3p are promising ITP prognosis predictors (El Zaiat et al., 2025). The expression levels of miR-200c-3p and miR-146a-5p are higher in pediatric chronic ITP than in newly diagnosed ITP, whereas lower miR-106b-5p levels were associated with complete response to therapy (El Zaiat et al., 2025). It is important to highlight that these findings relate to circulating miRNAs and do not establish whether these molecules are specifically associated with EVs.

Several studies have shown that EV-miRNAs offer potential advantages over other types of biomarkers for several reasons, such as stability, since the encapsulation protects miRNAs from RNase degradation, and accessibility through a simple blood sample, making non-invasive monitoring possible (Xu et al., 2022; Arailym et al., 2026). Interestingly, AGO2-miRNAs represent the majority of free plasma miRNAs, but they are less specific to their originating cell than EV-miRNAs (Groot and Lee, 2020; Geekiyanage et al., 2020). Despite their potential, the use of EV-miRNAs as biomarkers faces significant challenges that require standardization. Firstly, EV isolation methods are still controversial: no universally accepted standard protocol for EV isolation, and different methods produce EV populations with varying compositions (Welsh et al., 2024). More generally, pre-analytical and methodological heterogeneity, including differences in sample type and processing, may contribute to variability in miRNA profiles across different ITP studies (Liu et al., 2026). Most current studies on miRNAs in ITP have been conducted on small cohorts. Multiple, multicenter, and prospective studies are needed to validate these potential biomarkers identified.

6 Conclusion

This review sheds light on the emerging interplay between miRNAs, EVs, and immune dysregulation in pediatric ITP. In this context, pediatric ITP shows unique biological and clinical characteristics, such as greater immunological plasticity and the ability to restore immune tolerance compared to adults, further emphasizing the importance of investigating EV-miRNAs. Notably, the immune system in childhood is characterized by dynamic changes in the Treg/Th17 balance, T cell maturation, and cytokine production, which are also reflected in miRNA expression profiles.

EVs derived from haematopoietic and immune cells, function as active mediators of intercellular communication by transporting functional miRNAs, which serve not only as biomarkers reflecting a particular cellular state, but as messengers capable of modulating biological processes. As the expression of several miRNAs, such as miR-155, miR-146a and miR-142, has been revealed to be altered in ITP and implicated in Treg/Th17 imbalance, increased inflammatory cytokine production and disrupted megakaryocytic differentiation, therapeutic targeting of their levels with antagomiRs or miRNA mimics could be an innovative approach for ITP treatment. The stability, accessibility, and tissue specificity of EV-miRNAs make them particularly advantageous as novel non-invasive diagnostic and prognostic biomarkers and therapeutic targets for ITP treatment.

Despite growing findings linking epigenetic mechanisms to ITP pathogenesis, EV-miRNAs profiles investigation in pediatric cohorts remain scarce. Most of the available mechanistic and therapeutic evidence originates from ITP in adults, from experimental animal models or from other inflammatory diseases and, therefore, may not be directly extrapolated to pediatric ITP. Future research should focus on large-scale, multicenter, prospective studies comparing EV-miRNAs with circulating free miRNAs in carefully characterized pediatric cohorts of patients with ITP, including both newly diagnosed and chronic patients. Integration of EV-miRNA profiles with disease progression, response to treatment and platelet count could help determine their further diagnostic and prognostic value. At the same time, a deeper understanding of the interplay between EV biology and miRNA profiling could facilitate the development of more accurate diagnostic tools and engineered EV-based therapeutic strategies aimed at restoring immune homeostasis in pediatric ITP.

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Footnotes

Edited by: Martina Calore, University of Padua, Italy

Reviewed by: Hassan Shah, Guangzhou Medical University, China

John W. Semple, Lund University, Sweden

Jacy Gameiro, Juiz de Fora Federal University, Brazil