cfDNA is a type of extracellular double-stranded deoxyribonucleic acid fragment that is released during cell death and can be detected in a range of bodily fluids, including serum, plasma, urine, cerebrospinal fluid, saliva, and bronchial lavage. Its concentration is typically very low under normal physiological conditions. Studies have demonstrated that cfDNA levels are elevated in individuals with cancer compared to healthy individuals, as well as in those with inflammatory disorders and tissue damage. Elevated cfDNA levels in patients may be related to heightened apoptosis, necrosis, and impaired clearance of cellular debris associated with the disease, positioning cfDNA a potential indicator of disease activity. Apart from concentration, additional facets of cfDNA such as the ratio of fragment length, methylation pattern, and alteration in gene profile, serve to track the condition of disease. cfDNA liquid biopsy, as a non-invasive diagnostic tool, is widely used in clinical practice today, including prenatal detection of fetal chromosomal abnormalities, cancer screening and phenotyping, and cardiovascular disease diagnosis (Pös et al., 2018; Ranucci, 2019; Dao et al., 2023). In the field of rheumatology, conditions such as Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), Systemic Sclerosis (SSc), and primary Sjögren’s syndrome (pSS) exhibit increased cfDNA levels (Vakrakou et al., 2017; Nandi et al., 2020), which correlate with disease activity in patients (Abdelal et al., 2016). This highlights the potential of cfDNA as a diagnostic indicator for a spectrum of rheumatic conditions. Advances in nucleic acid testing technology and the understanding of DNA receptors’ roles in inflammation and rheumatic diseases have further propelled cfDNA research (Figure 1). This review aims to provide a detailed introduction to cfDNA and summarize its ability to reflect disease status changes, thereby deepening understanding of cfDNA in rheumatic diseases.

cfDNA fragments range from 70 to 200 base pairs (bp) in length (Cicchillitti et al., 2017). The size of these molecules depends on the mechanisms of their fragmentation. Typically, the peak length is around 166 bp, corresponding to DNA fragments associated with nucleosomes (Snyder et al., 2016). cfDNA can also form complexes by binding with certain proteins and membrane structures.

RA, an autoimmune disorder, is marked by persistent synovial inflammation and advancing joint harm, potentially resulting in joint abnormalities, functional loss, and a significant incidence of disability and deformities (Weyand and Goronzy, 2021). Timely and effective treatment is crucial for controlling inflammation, preventing disease progression, functional impairment, and adverse outcomes. The detection of Rheumatoid Factor (RF) and Anti-Citrullinated Peptide Antibodies (ACPA) shows good sensitivity and specificity in the early diagnosis of RA, but autoantibodies may share epitopes, and some non-RA rheumatic diseases or chronic infections may also present with positive RF or anti-CCP antibodies (Taylor et al., 2011). Some RA patients may also present with negative RF and anti-CCP antibody results (Jang et al., 2022). Serological diagnosis may have false negatives and false positives. Moreover, the diverse pathogenesis of RA leads to significant patient heterogeneity, and some patients may not respond well to treatment. Therefore, suitable biomarkers need to be developed to optimize serological diagnosis and guide clinical treatment strategies (Zhao and Li, 2018).cfDNA, which has garnered attention in oncology, is an ideal candidate for developing new biomarkers.

Research indicates increased levels of cfDNA in the peripheral blood and synovial fluid in patients with RA. Dong et al. evaluated the levels of cfDNA in the plasma and synovial fluid of 80 patients with RA and a control group, utilizing Picogreen technology. The findings showed a significant difference in plasma cfDNA concentrations between individuals with RA and those without. Unexpectedly, the average concentration of cfDNA in the synovial fluid of patients with RA was about 77 times greater than that in plasma (Dong et al., 2020). Moreover, cfDNA concentration correlates with RA disease activity. Rykova et al. found that RA patients exhibiting greater disease activity showed elevated median levels of plasma circulating nDNA and CRP, in contrast to those with lower disease activity and the control group (Rykova et al., 2017). And, following the application of biological Disease-modifying Anti-rheumatic Drugs (bDMARDs), a clear positive correlation emerged between plasma cfDNA concentration and DAS28 scores in RA (Macáková et al., 2022). However, the correlation between synovial fluid cfDNA and plasma CRP was not strong (Birkelund et al., 2020). Also, Macáková et al. analyzed the relationship between RF, ACPA concentrations, and cfDNA, finding no significant correlation. This implies that cfDNA might depend on different inflammatory mechanism. Combining these biomarkers could potentially enhance the serological diagnosis of RA.

RA synovial fluid contains a notably greater amount of cfDNA compared to plasma, indicating that the release of cfDNA in RA mainly occurs in the joints and is probably linked to inflammation (Dong et al., 2020). Compared to other types of arthritis, RA synovial fluid cfDNA levels are 38.8 times higher than in osteoarthritis patients, while there is no statistically significant difference in intra-articular cfDNA levels between RA and spondyloarthritis patients (Birkelund et al., 2020). Analysis of the protein composition in RA synovial fluid reveals that neutrophil-derived proteins are predominant. This finding is consistent with RA’s pathogenesis, as there is a significant infiltration of neutrophils in the synovial fluid of RA patients. Furthermore, NETs-related proteins from neutrophils are associated with synovial cfDNA levels. These observations suggest that cfDNA in the synovial fluid predominantly originates from NETosis. The intense neutrophil-mediated inflammation and NETosis contribute to the high levels of cfDNA observed in RA synovial fluid (Wright et al., 2021) (Figure 3). In turn, high levels of cfDNA in synovial fluid could serve as a marker for neutrophil death and NETs formation within the joint. The quantification of synovial cfDNA therefore may be utilized as a diagnostic tool for severe arthritis and potentially help in identifying and monitoring the extent of inflammation in RA.

To further clarify the pathological relevance of synovial cfDNA, Dong et al. isolated and collected synovial cfDNA from RA patients and used it to stimulate inflammatory cells in vitro (Dong et al., 2020). Flow cytometry analysis revealed an increase in inflammatory cytokine expression in inflammatory cells following stimulation with synovial cfDNA. Additionally, it was discovered that serum from patients with RA triggers Gasdermin D (GSDMD)-dependent pyroptosis in monocytes, linked to increased cfDNA levels in the peripheral blood (Wu et al., 2020). The findings validate the inflammatory properties of cfDNA in RA. And the inflammatory properties of cfDNA in RA are attributed to its low-methylated CpG sequences. Through genomic sequencing of the synovial cfDNA in patients, it was found that, compared to OA patients, cfDNA from RA patients has a higher density of CpG sequences, with the methylation level of CpG sequences significantly lower than that in OA patients. The TLR9 receptor primarily identifies these inflammatory low-methylated CpG sequences as ligands, which exert a strong inflammatory impact. In cells stimulated by cfDNA from RA, NF-κB nuclear translocation induced by triggering of the TLR9 pathway could be observed (Hashimoto et al., 2023). Cell analysis shows that RA CpG-motif-rich (CMR) sequence has a potent ability to activate inflammatory cells to produce the inflammatory mediator TNF-α, while using smallinterferingRNA (siRNA) to suppress TLR-9 markedly reduces the effect of CMRs in inducing TNF-α.

Abnormal release of cfDNA activates innate immune pathways such as TLR-9, leading to chronic inflammation and promoting the advancement of RA. Clearing excess cfDNA could be effective in reducing inflammation and lessening the severity of RA. The innovative RA therapy strategy targeting cfDNA involves administering cationic nanoparticles into the body, using charge interactions to attach to the negatively charged cfDNA, thus removing surplus cfDNA and suppressing immune response (Chen and Tsokos, 2022; Peng et al., 2024; Liu, 2003). As scavengers of DNA molecules, cationic nanoparticles markedly reduced arthritis symptoms in rats; yet, their potent positive charge could lead to systemic toxicity in intravenous blood flow, and their effectiveness is hindered by biocompatibility, serum tolerance, and subpar clearance. For this, researchers altered the cationic nanoparticles chemically to precisely manipulate their molecular composition, with the goal of harmonizing toxicity, enhancing molecular binding capacity, and boosting biodistribution. In the Collagen-Induced Arthritis (CIA) rat model, the modified cationic dendritic polymers successfully reduced symptoms like joint inflammation, increased synovial growth, and bone damage.

On the other hand, cfDNA might serve as a marker to predict the treatment response of bDMARDs in RA. Previously noted, patients with RA exhibit increased levels of cfDNA in their blood. However, Dunaeva’s research revealed a contrasting result, showing that the serum cfDNA concentrations in patients with early-stage RA are comparable to those in healthy individuals, whereas in patients with late-stage RA, serum cfDNA levels are markedly less than those in early-stage RA patients and healthy subjects (Dunaeva et al., 2015). The authors asserted that the fluctuations in cfDNA levels are not caused by variations in DNase activity and hypothesized that the decrease in cfDNA levels in RA patients may be related to the treatment they received. Indeed, the use of medicine can affect cfDNA levels, with report indicating that in 7 out of 10 patients, plasma cfDNA levels changed significantly (either rising or falling) within an hour after infliximab infusion (Zhong et al., 2007). The findings of Hashimoto’s study reveal that RA patients undergo alterations in cfDNA levels post-treatment with bDMARDs, linked to improved disease activity (Hashimoto et al., 2017). Subsequent studies have confirmed that bDMARDs like tocilizumab may possess strong DNase activity, directly degrading cfDNA and thereby inhibiting the inflammatory response induced by the TLR-9 pathway (Hashimoto et al., 2023). To analyze the impact of bDMARDs on cfDNA in RA patients and clarify the correlation between cfDNA level changes and treatment response, Macáková et al. collected blood samples from RA patients before starting bDMARDs treatment, as well as at 3 months and 12 months after treatment initiation. The data showed that, after 3 months of bDMARDs treatment, extracellular DNA in responders decreased significantly by 60%, with extracellular-nDNA decreasing by 58%. In contrast, plasma extracellular DNA levels remained unchanged in the moderate response and non-responding groups (Macáková et al., 2022). Macáková holds the view that plasma extracellular DNA, particularly nuclear-derived extracellular DNA, has the potential to monitor RA treatment response. Conversely, Rykova and Laukova possess different views. Rykova found no significant correlation between the plasma cfDNA of RA patients and the duration of treatment after the adoption of a unified therapy combining methotrexate, etoricoxib, and folic acid. Another group of patients received combined treatment with rituximab and methotrexate, and there was no difference in cfDNA levels between the two treatment groups (Rykova et al., 2017). The findings of Laukova’s study revealed a 3-month postponement in the decrease of plasma cfDNA in RA sufferers after bDMARD treatment, in contrast to the clear improvement in clinical manifestations and laboratory parameters. Consequently, Laukova and colleagues are of the opinion that cfDNA does not serve as a reliable indicator of disease activity (Lauková et al., 2018). Due to variations in cfDNA detection intervals, patient health histories, and therapeutic medications, a direct comparison of the findings is not feasible. Post-treatment cfDNA levels may be influenced by various factors, including the timing of blood draws and the types of bDMARDs used. Therefore, to understand the relationship between cfDNA level changes and responses to different bDMARDs treatments, long-term dynamic monitoring and the inclusion of more trial samples are necessary.

To summarize, RA patients frequently exhibit increased cfDNA levels, which appear to correlate with the disease’s progression and the effectiveness of treatment (see summary in Table 1). Consequently, cfDNA could be an effective parameter for tracking RA’s disease progression and forecasting future treatment outcomes. There’s no significant connection between cfDNA and the levels of serological markers, likely because they depend on separate inflammatory mechanisms. However, cfDNA could bridge the diagnostic divide for patients with seronegative RA, regardless of serological factors. Large-scale longitudinal research remains essential to document the evolving alterations of cfDNA in RA throughout the disease’s development and following treatment. Additionally, while clearing excess cfDNA can significantly alleviate joint inflammation, further optimization and testing are required before cfDNA can be implemented in clinical practice. It is anticipated that cfDNA will soon become a new tool in the clinical management of RA.

Patients with SLE exhibit elevated cfDNA levels in contrast to healthy individuals, yet the link between cfDNA concentrations and SLE disease progression is still debated. The majority of research indicates a direct correlation between cfDNA amounts and SLE disease activity. To determine the correlation between SLE disease activity and cfDNA levels, Xu et al. compared cfDNA levels between 18 patients with high SLEDAI scores and 40 patients with low SLEDAI scores. They found that the median cfDNA levels were significantly higher in patients with active disease compared to those with inactive disease. Additionally, there was a noticeable pattern in which elevated cfDNA levels correlated with increased SLEDAI scores among all patients, including those who were not pregnant and those who were (Xu et al., 2018). However, Atamaniuk and Tug found no correlation between cfDNA levels and SLEDAI scores in their studies (Tug et al., 2014; Atamaniuk et al., 2011). Despite this, a series of plasma measurements in SLE patients suggest that fluctuations in cfDNA levels can partially reflect changes in disease status, particularly disease progression. Indeed, studies have reported conflicting results regarding the relationship between cfDNA and inflammatory markers like CRP, as well as complement components C3 and C4. Some research indicates a correlation, while others find no significant link (Tug et al., 2014; Xu et al., 2018; Hendy et al., 2016). Xu et al. further investigated whether the inflammatory process was associated with higher cfDNA levels by examining other inflammatory markers. They found that reductions in C3 and C4 levels were not associated with increases in cfDNA levels. In Tug’s study of the relationship between cfDNA levels and individual clinical parameters, it was found that cfDNA levels showed a significant correlation with C3 and C4 in both SLE patients and healthy controls. Additionally, in the healthy control group, cfDNA was significantly correlated with CRP, whereas no such correlation was observed in SLE patients. In Hendy’s correlation study, cfDNA levels were significantly negatively correlated with C3, positively correlated with serum CRP levels, and showed no correlation with age, sex, SLEDAI score, or C4. These conflicting results may be due to differences in methodologies and clinical diversity among patient populations. The levels of C3, C4, and CRP can vary over time, so results may differ based on the timing of assessments. Although the results are not uniform, cfDNA levels in SLE show a strong correlation with medical assessments. Elevated cfDNA levels reflect the inflammatory state, suggesting that cfDNA could potentially be considered an acute-phase reactant in SLE.

Aside from inflammation markers, cfDNA levels are also correlated with autoantibody titers. Atamaniuk’s study found a statistically significant correlation between cfDNA and anti-histone antibodies in SLE patients, while Hendy discovered that cfDNA levels were associated with the disease severity marker, anti-nucleosome antibodies. On the other hand, it is unexpected that Hendy’s correlation analysis found a negative correlation between the levels of cfDNA and ANA, anti-dsDNA levels, whereas Xu did not find a correlation between dsDNA antibodies and cfDNA (Atamaniuk et al., 2011; Xu et al., 2018; Tug et al., 2014). In theory, cfDNA is likely the initiating factor for the production of anti-dsDNA in SLE, and there should be a certain positive correlation between the two. For this anomalous phenomenon, researchers thought that in patients with high autoantibody titers, the formation of DNA-antibody immune complexes may directly interfere with cfDNA detection or accelerate the clearance of DNA-antibody complexes (Truszewska et al., 2017). Continuous sampling might provide a better understanding of the association between cfDNA levels and anti-dsDNA antibody levels.

Apart from differences in levels, the cfDNA molecular characteristics in SLE patients also differ from those in healthy individuals (Chan et al., 2014). Rebecca W. Y. Chan conducted a high-resolution analysis of the biological characteristics of plasma DNA in SLE patients using large-scale parallel genomics and methylomics sequencing, and discovered numerous differences between the plasma DNA of SLE patients and those of healthy individuals. Firstly, the data obtained from sequencing studies indicated that the cfDNA profiles in SLE patients contained multiple measured genomic representations (MGRs) that were either under-expressed or over-expressed, which were not present in healthy controls. Researchers hypothesize that in SLE, anti-DNA antibodies may preferentially bind to specific cfDNA sequences to form complexes, with the cfDNA protected in these antibody-bound regions, leading to an increase in genomic representations in certain areas and a decrease in those not preferentially bound by antibodies, resulting in the emergence of abnormal MGRs. This phenomenon has currently only been observed in SLE, and the significance of the abnormal MGRs in SLE remains unclear. Secondly, the distribution of cfDNA fragment lengths varies between patients with SLE and healthy individuals. In healthy individuals, the plasma cfDNA molecular size is predominantly centered around 166 bp, with a prominent peak at this length in the cfDNA size distribution curve (Shi et al., 2020).In contrast, SLE patients exhibit a reduced peak height at 166 bp, indicating a lower abundance of cfDNA fragments of this size. Moreover, there is an increase in the peak height of smaller cfDNA fragments, particularly those shorter than 115 bp. These changes are more pronounced in patients with active SLE compared to those with inactive SLE. In further research, Rebecca W. Y. Chan defined plasma DNA molecules ≤115 bp as short DNA fragments and measured the percentage of short plasma DNA fragments in each sample. The median percentage of short plasma DNA fragments in the plasma was 10% for healthy individuals, 14% for patients with inactive SLE, and 31% for patients with active SLE. The percentage of short DNA fragments showed a positive correlation with SLEDAI scores and anti-dsDNA antibody levels.

In addition, cfDNA is hypomethylated in SLE. The genome-wide methylation density is significantly lower in patients with active SLE compared to healthy individuals and those with inactive SLE. Active SLE patients have a greater number of hypomethylated regions than those in the inactive group. There is a correlation between the percentage of hypomethylated regions and both SLEDAI scores and anti-dsDNA antibody levels. Research reveals that injecting hypomethylated homologous apoptotic DNA can trigger SLE-like symptoms and cause non-susceptible mice to develop proteinuria and anti-DNA antibodies, indicating that hypomethylated DNA has immunogenic properties and is related to the pathogenesis of SLE (Wen et al., 2007). It is hypothesized that cfDNA hypomethylation may influence disease progression by inducing excessive production of anti-DNA antibodies. Notably, short DNA fragments in the plasma of SLE patients have even lower methylation levels. There is a negative correlation between genome-wide methylation density and the proportion of short DNA fragments (≤115 bp). The decrease in methylation density is more pronounced in the active SLE group compared to healthy individuals and those with inactive SLE, with plasma DNA fragments progressively shortening.

Hypomethylation of DNA plays a crucial role in the molecular process of SLE, with the stable intermediate product of active DNA demethylation, 5-Hydroxymethylome (5hmC), influencing gene expression through gene modification, contributing equally to the development of SLE. In order to explore the 5hmC modification status in cfDNA from SLE patients, Tong et al. conducted bioinformatic analysis of the whole-genome 5hmC profile of plasma cfDNA from healthy controls and SLE patients to identify differential hydroxymethylated regions (DhMR) (Tong et al., 2024). The results showed significant differences in both enrichment and motifs between the plasma cfDNA 5hmC profiles of the SLE and HC groups. Functional enrichment annotation analysis was then performed to further explore the biological processes involved in 5hmC modification in SLE. Compared to the healthy control group, genes related to metabolic and immune pathways in SLE patients exhibited dysregulated 5hmC enrichment, including the Wnt signaling pathway, inflammatory responses, and cytokine-mediated signaling pathways. cfDNA with high DhMRs may be closely associated with SLE. Based on this, the experiment additionally included a group of SLE patients with higher SLEDAI scores to investigate the relationship between 5hmC and disease progression. Compared to the original SLE and HC groups, this subgroup exhibited higher insertion fragment size and multiple enrichment frequencies. The increase in frequency in this subgroup confirms that the level of 5hmC rises with SLE activity, indicating a significant difference in the distribution of 5hmC during disease progression. The above conclusion highlights the potential of 5hmC modifications in cfDNA as a candidate biomarker for the diagnosis and monitoring of SLE.

The differences in cfDNA molecular characteristics between healthy individuals and SLE patients suggest variations in cfDNA sources. Jeremic found that SLE patients have higher levels of NETs-related markers compared to healthy controls. NETs are abnormally increased in SLE patients and become a major source of cfDNA (Jeremic et al., 2019). Low-density granulocytes (LDGs) are a subset of neutrophils with enhanced NETs formation ability. Henning found that, compared to healthy controls, SLE patients have a significantly higher proportion of LDGs and elevated levels of circulating plasma NETs (Henning et al., 2024).In SLE, elevated cfDNA levels may also be due to impaired NETs clearance. Leffler found that patients with active SLE have reduced NETs degradation ability (Leffler et al., 2012). NETs are composed of extracellular chromatin, and their accumulation is primarily regulated by the degradation activity of DNase I and DNase IL3 (Angeletti et al., 2021). A mutation in one of the DNase genes can lead to the formation of anti-DNA antibodies and is associated with severe lupus-like syndromes and lupus nephritis. In SLE, DNase may have inactivating mutations, reducing its activity compared to healthy individuals (Yasutomo et al., 2001). Relevant studies in mouse models also show that mice lacking DNase I display typical symptoms of SLE, including the production of ANA and the accumulation of glomerular immune complexes (Napirei et al., 2000). Additionally, anti-DNase antibodies can impair DNase function. Hartl’s research indicates that reduced DNase IL3 activity in Lupus Nephritis (LN) patients is linked to neutralizing anti-DNase IL3 antibodies, which contribute to increased cfDNA accumulation and anti-DNA autoimmunity in SLE (Hartl et al., 2021). Notably, the lack of DNase correlates with alterations in the jagged ends of cfDNA, and a shortage of DNase IL3 can markedly lower the jagged index in plasma DNA exceeding 166 bp. Studies indicate that in patients with SLE, the DNA’s molecular jagged index exceeding 200 bp is less than that in healthy individuals, and this reduction is more noticeable in those who are active. The potential application of this molecular characteristic could may potentially be used for the differential diagnosis of SLE (Ding et al., 2022).

Inefficient DNA degradation is associated with renal involvement. Previous studies have noted the relationship between plasma cfDNA and LN, but the analysis of cfDNA in LN patients is still in its early stages. Recently, Wang et al. described the characteristics of cfDNA in LN patients. They found that the concentration of plasma cfDNA in LN patients was significantly lower than that in non-LN patients. Moreover, the proportion of short cfDNA fragments was notably higher in LN patients compared to non-LN patients. There was also a significant difference in the average methylation scores between LN patients and non-LN patients (Wang et al., 2022). The lower cfDNA concentration in LN patients may be associated with the presence of anti-dsDNA antibodies. Regarding the correlation between cfDNA and LN disease activity, Zhang et al. found that the average cfDNA concentration was significantly higher in patients with active LN compared to those with inactive LN. Additionally, there was a significant correlation between cfDNA concentration and parameters related to the severity of LN. In the SLE group, cfDNA concentration was positively correlated with 24-h urine protein levels and negatively correlated with albumin levels and endogenous creatinine clearance. These data suggest that elevated cfDNA levels may be associated with LN disease activity (Zhang S. et al., 2014). Compared to the aforementioned studies, Truszewska et al. conducted a more detailed validation of the hypothesis that cfDNA profiles are related to LN clinical features, proposing the use of cfDNA levels, the intracellular mtDNA/cf-mtDNA ratio, the cfDNA fragmentation index, and the presence of 54-149 bp and 209-297 bp fragments as clinically relevant parameters for characterizing cfDNA profiles (Truszewska et al., 2020). Research revealed that SLE patients had an average intracellular mt/cf-mtDNA ratio triple that of healthy individuals, and those with a higher mt/cf-mtDNA ratio had a relative cf-mtDNA copy number linked to proteinuria surpassing 0.5 g/day and LN. The fragmentation index reflects cfDNA integrity, and the results showed that SLE patients had significantly higher fragmentation indices compared to healthy individuals, with a higher fragmentation index associated with a lower estimated Glomerular Filtration Rate (eGFR) in SLE patients. Patients with Class V LN had higher fragmentation indices than those with other types of LN. A variance was observed in the distribution of cfDNA sizes between patients with SLE and healthy controls, where about 30% exhibited fragments ranging from 54 to 149 bp and 209-297 bp, absent in healthy subjects. In addition, patients possessing fragments of 54-149 bp and 209-297 bp exhibit notably elevated fragmentation indices and increased cfDNA concentrations. Further analysis revealed that patients with 209-297 bp fragments have lower eGFR, while the concentration of the 54-149 bp fragment is positively correlated with anti-dsDNA antibody titers, CRP, and SLEDAI. The results suggest that the disruption of the relationship between intracellular and cf-mtDNA, the cfDNA fragmentation index, and the presence of the 54-149 bp and 209-297 bp fragments may be related to disease activity and impaired kidney function, and thus should be considered as parameters for characterizing the cfDNA profile. The maximum observed values of the aforementioned parameters in healthy subjects serve as the threshold for choosing patients whose cfDNA profile mirrors that of healthy persons. Patients with a healthy-like cfDNA profile exhibit significantly higher eGFRs than other patients and show no signs of active arthritis. Such patients do not have indications for kidney biopsy or are diagnosed with class II, III, or II + III LN, whereas those without this profile are diagnosed with class IV, V, or VI LN (including those with types combined with class IV or V) or are kidney transplant recipients. SLE patients whose cfDNA levels, mt/cf-mtDNA ratio, or fragmentation rate exceed twice the upper limit of the healthy cfDNA group and contain fragments of 54-149 bp or 209-297 bp are classified into a distinct cfDNA profile group. Patients with different cfDNA profiles have significantly lower eGFRs, with nearly 74% of these patients diagnosed with class IV, V, or VI LN or being kidney transplant recipients, and none being diagnosed with class II, III, or II + III LN. Therefore, the results indicate that the type of lupus nephritis is associated with the cfDNA profile, and lupus nephritis with a poorer prognosis is clearly linked to a distinct cfDNA profile.

cfDNA is involved in the pathogenesis of SLE and is associated with renal involvement (see summary in Table 2). In SLE patients, cfDNA levels are elevated and fluctuate with disease status and some degree of treatment intervention. However, the association between cfDNA and disease activity or inflammatory markers has only been partially confirmed. The formation of immune complexes may affect data measurement, leading to result deviations. Therefore, whether cfDNA concentration can be used to assess SLE disease activity remains under question. On the other hand, studies have shown specific molecular features of cfDNA in SLE, and its apparent genetic distribution may serve as a new clinical marker for SLE. The mechanisms of cfDNA in SLE warrant further exploration.

Ankylosing spondylitis (AS) is characterized by progressive chronic inflammation of the axial joints and surrounding tissues. In late stages, it can lead to ankylosis of the spinal joints, reduced spinal mobility, and severe disability (Ebrahimiadib et al., 2021). Under genetic conditions associated with AS, persistent micro-injuries and chronic inflammation that promote disease progression can lead to increased release of cfDNA into the circulation (Mauro et al., 2021). Therefore, cfDNA may serve as a potential biomarker for assessing disease activity in AS. Peng et al. conducted an in-depth exploration of circulating cfDNA in AS patients and its relationship with clinical disease activity and treatment response (Peng et al., 2024). The study found that cfDNA levels in AS patients were significantly higher than in healthy controls and positively correlated with CRP, Ankylosing Spondylitis Disease Activity Score (ASDAS)-CRP, and neutrophil counts. The use of Nonsteroidal Antiinflammatory Drugs (NSAIDs) in combination with DMARDs or tumor necrosis factor inhibitors can reduce cfDNA levels, and a decrease in cfDNA levels after treatment is associated with a good therapeutic response. Notably, treatment-naive patients with higher cfDNA levels respond better to combined therapy than to NSAIDs alone, while those with lower cfDNA levels show similar response rates to either combined or single NSAIDs treatment. In summary, circulating cfDNA levels are significantly correlated with disease activity and treatment outcomes in AS patients. Additionally, cfDNA levels in treatment-naive patients can predict prognosis for different treatments, making cfDNA an effective biomarker for AS inflammation.

NETs are considered to be closely associated with the pathogenesis of AAV. In this process, ANCA plays a key role in triggering NETs formation, which leads to the release of mtDNA and nDNA, thereby inducing inflammatory response. In last year, Karlsson has conducted an in-depth exploration of the differences in circulating cfDNA levels in AAV patients during active and remission phases (Karlsson et al., 2023). Compared to the remission phase, there is no significant difference in mtDNA or nDNA levels in patients with active disease; nor is there a difference in the ratio of mtDNA to nDNA. Moreover, cfDNA shows no correlation with age, gender, clinical phenotype, ANCA positivity, renal involvement, or Birmingham Vasculitis Activity Score (BVAS). Considering the limited number of participants in this research (16 AAV patients), the lack of correlation between cfDNA and disease activity could be influenced by bias. Conversely, Alam’s research encompassed specimens from 103 individuals suffering from ANCA-related vasculitis (33 in active disease, 70 in remission) alongside 41 non-affected subjects. Research revealed that active AAV patients had elevated plasma mtDNA levels compared to healthy individuals and those in remission, while the nDNA levels in active patients were comparable to those in remission. And a notable increase in the mtDNA/nDNA ratio could be observed among active patients in contrast to those in remission (Alam et al., 2019). Within AAV, there’s an increase in cfDNA levels, and its makeup varies depending on the activity of the disease. During active illness, mtDNA, as opposed to nDNA, shows an increase, possibly due to mtDNA being released during platelet activation and the variance in its clearance compared to nDNA. A subsequent study by Giaglis indicated that mtDNA was associated with BVAS and, in multivariate analysis, correlated with AAV activity. In 27 AAV follow-up patients, alterations in mtDNA correlated with shifts in BVAS, in contrast to CRP or ANCA titers, which demonstrated no link to BVAS variations (Giaglis et al., 2023). Both studies confirm the superiority of mtDNA over nDNA in monitoring AAV activity. However, the application of the mtDNA/nDNA ratio measurement in clinical practice still needs to be clarified.

Similar to SLE, there is an abnormality in NETs metabolism in Idiopathic Inflammatory Myopathies (IIMs) (W. Ma et al., 2023). To verify the pathogenic role of NETs in Dermatomyositis (DM) and Polymyositis (PM), Zhang et al. tested the ability to induce and degrade NETs in plasma samples from 97 DM/PM patients (72 with DM, 25 with PM) and 54 healthy controls. The study found a notable increase in plasma cfDNA levels in IIMs patients compared to healthy individuals, with ILD patients showing higher cfDNA concentrations than those without ILD. Consistent with the cfDNA results,DM/PM patients exhibit a markedly improved capacity to trigger NETs compared to the control group, alongside a significant decrease in degradation capacity and DNase I activity. Notably, patients with concurrent Interstitial Lung Disease (ILD) had even lower DNase I activity and the poorest NETs degradation capacity. In IIMs patients, particularly those with ILD, the reduced DNase I activity means that excessive NETs are not fully degraded, suggesting that abnormal NETs regulation may be involved in the pathogenesis of IIMs and could be a contributing factor to the onset and exacerbation of ILD (Zhang S. et al., 2014). According to reports, plasma DNA can induce the production of pro-inflammatory cytokines by activating the TLR-9 and cGAS/STING signaling pathways, thereby causing lung injury (Zhang et al., 2016; Ryu et al., 2020; Ma et al., 2020). cfDNA, as a significant component of NETs and pro-inflammatory DAMPs, may play a role in the development of ILD at IIMs. These findings suggest that cfDNA is involved in the pathogenesis of IIMs, but the specific relationship between cfDNA and disease activity in IIMs still needs further exploration.

cfDNA, being a non-intrusive liquid biopsy technique, is somewhat indicative of the activity and treatment efficacy in rheumatic conditions. Nonetheless, numerous problems remain that require attention. Initially, the precise mechanisms behind the release of cfDNA are yet to be clarified. Pinpointing the origins of cfDNA in different rheumatic conditions is crucial for comprehending its application in diagnosing, predicting, or tracking these illnesses. Secondly, cfDNA levels are affected by multiple factors, and a single quantitative test might not be precise for disease diagnosis. In different physiological and pathological states, not only does the level of cfDNA change, but molecular characteristics such as methylation density, gene lineage, fragment size distribution, and others also vary. For example, ctDNA contains tumor mutation genes, so it helps assess tumor progression, prognosis, and aids in targeted therapy. To apply cfDNA diagnostics in the clinical treatment of rheumatic and immune diseases, we need to focus on related qualitative research findings of cfDNA. The genetic profiling of cfDNA in rheumatic diseases will contribute to the realization of personalized precision medicine. Additionally, there is an obvious inconsistency in the types of samples, their processing techniques, cfDNA extraction, cfDNA quantification, and the way cfDNA quantification outcomes are presented and interpreted. And with the emergence of qualitative studies on cfDNA, its complexity has also increased. Crucially, present studies predominantly concentrate on RA and SLE, with limited research on other rheumatic conditions and a scarcity of data. The absence of extended-duration studies and constraints in sample sizes compromise the dependability of the findings. A comprehensive grasp of cfDNA’s role as a biomarker in autoimmune disorders necessitates a methodical scientific structure and joint endeavors in executing extensive, multi-institutional forward-looking trials.