The diagnosis of RA is typically established through a comprehensive evaluation encompassing a detailed analysis of the patient's medical history, a thorough physical examination, and the evaluation of pertinent laboratory test results. However, these aforementioned diagnostic techniques exhibit several limitations. For example, gathering clinical information often remains subjective, reliant on the patient's descriptions and the physician's interpretation. This can lead to discrepancies in the diagnosis of the same patient when seen by different medical practitioners. Early symptoms of RA, such as joint pain, swelling, and stiffness, often lack specificity, resembling manifestations of other disorders, posing a potential challenge in the timely diagnosis and management of RA in its initial stages. Although imaging techniques like radiography, MRI and ultrasound technology are capable of assessing joint structure and pathology, their sensitivity in detecting early RA without evident bone damage is comparatively inadequate[22–25]. The most robust blood-based assays for RA currently heavily rely on serum protein biomarkers, with ACPA and RF being pivotal components of the standard RA classification criteria set forth by the American College of Rheumatology and the European League Against Rheumatism [26]. However, RF's specificity in RA diagnosis is relatively limited, as elevated RF levels can also manifest in numerous other medical conditions [27]. Despite the favorable balance of sensitivity and specificity demonstrated by ACPA in diagnosing RA, a clinical challenge persists, as approximately 33% of RA patients exhibit negative serum ACPA results[28].
In an effort to address this critical clinical concern, Hu et al. discovered a substantial elevation in serum levels of soluble scavenger receptor A (sSR-A) in cases of rheumatoid arthritis (RA). They identified sSR-A as a potential diagnostic marker for RA, particularly in instances of early and seronegative RA, as demonstrated through an extensive multicenter trial [29]. Li et al. employed human protein microarray technology to successfully screen two novel autoantibodies of anti-PTX3 and anti-DUSP11, which can serve as novel diagnostic biomarkers for ACPA-negative RA[30]. However, the aforementioned studies have primarily focused on protein-based biomarkers. Protein markers are subject to intricate regulatory mechanisms, encompassing post-translational modifications and protein interactions. These complexities render their precise and sensitive detection and quantification challenging, particularly in the initial phases of a disease [31, 32]. On the contrary, RNA markers can detect early changes in cells or tissues and are often more reversible, rendering them advantageous for assessing treatment efficacy and monitoring disease progression [33]. Our previous study also demonstrated that transfer RNA-derived small RNAs (tsRNAs) derived from exosomes exhibit distinct expression patterns in the blood and urine of SLE patients, implicating their involvement in immune regulation and inflammatory pathways [34, 35]. Similarly, in this study, we have, for the first time, established that exosome-derived miRNAs, including miR-885-5p and miR-6894-3p, exhibit significant differences in diagnosis between healthy control subjects and patients with RA, both with ACPA-positive and ACPA-negative conditions. Our findings imply that miR-885-5p and miR-6894-3p derived from exosomes may become a more general and comprehensive biomarker of RA that could applied in the diagnosis and identification of RA. Moreover, exosome derived miR-885-5p has a strong positive correlation with laboratory indicators of RA include ACPA and MCV, suggesting a potential role for miRNAs in RA disease activity and progression. We also examined the specificity of candidate miRNAs derived from exosomes and found that miR-6894-3p was upregulated in RA when compared to other autoimmune diseases, with good discriminatory capacity.
In order to further validate the potential value of exosome-derived miRNAs for therapeutic monitoring, we conducted a dynamic monitoring of miRNA levels in 9 RA patients undergoing MTX treatment. MTX stands as the most classical therapeutic option for rheumatoid arthritis, highly endorsed by guidelines worldwide as the preferred monotherapy. The dynamic assessment of MTX's effectiveness assumes a pivotal role in empowering rheumatologists to promptly adjust treatment strategies, thereby optimizing therapeutic outcomes and diminishing the risks of disease activity and joint damage for patients [36]. Thus, we aimed to assess the expression levels of serum exosome-derived miR-885-5p, miR-6894-3p, and miR-1268a in nine adult patients with active RA undergoing stable doses of methotrexate MTX. The outcomes revealed a progressive reduction in the expression of these miRNAs on day 29 following treatment. Furthermore, notable declines were observed in parameters including TJC28, SJC28, DAS28-CRP, and DAS28-ESR, coinciding with an amelioration of patient symptoms. These findings underscore the potential utility of exosome-derived miRNAs as valuable adjuncts for rheumatologists in gauging the efficacy of MTX in the treatment of RA.
Finally, a bioinformatics analysis was performed to further predict the potential involvement of the candidate miRNAs in the pathogenesis of RA and identify their target genes. The KEGG analysis revealed that these three candidate miRNAs were primarily associated with the mTOR signaling pathway and the MAPK signaling pathway which play a pivotal role in the occurrence and progression of rheumatoid arthritis[37, 38]. Furthermore, our investigations unveiled a noteworthy enrichment of these key miRNAs within signaling pathways associated with "Herpes simplex virus 1 infection." Herpes simplex virus type 1 (HSV-1) is primarily associated with infections in certain immunosuppressed patients, and recurrent HSV-1 infections can manifest with increased aggressiveness [39]. Indeed, recent research suggests that prolonged treatment of RA with tofacitinib may elevate the risk of encephalitis resulting from HSV-1 reactivation, thereby posing an augmented risk of severe and life-threatening illness [40]. Furthermore, in a case report of a patient with dermatomyositis treated with MTX, severe oral inflammation was triggered by reactivation of HSV-1[41]. The reactivation of HSV-1 triggers a range of antiviral responses, potentially leading to the disruption of immune homeostasis [42]. An increasing number of researchers are displaying heightened interest in investigating the effects of viruses on autoimmune diseases [43], thereby opening up new avenues for future research into the underlying mechanisms of autoimmune disorders.