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Increased Circulating Cell-Free DNA in Eosinophilic Granulomatosis With Polyangiitis: Implications for Eosinophil
Teppei Hashimoto1, Shigeharu Ueki2, Yosuke Kamide3
1Division of Diabetes, Endocrinology and Clinical Immunology, Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya, Japan.
Background:
Endogenous DNA derived from nuclei or mitochondria is released into the blood circulation as cell-free DNA (cfDNA) following cell damage or death. cfDNA is associated with various pathological conditions; however, its clinical significance in antineutrophil cytoplasmic antibody-associated vasculitis (AAV) remains unclear. This study aimed to evaluate the clinical significance of cfDNA in AAV.
Methods:
We enrolled 35 patients with AAV, including 10 with eosinophilic granulomatosis with polyangiitis (EGPA), 13 with microscopic polyangiitis, and 12 with granulomatosis with polyangiitis. Serum cf-nuclear DNA (cf-nDNA) and cf-mitochondrial DNA (cf-mtDNA) levels were measured by quantitative polymerase chain reaction before and after the initiation of immunosuppressive therapy. Tissue samples from EGPA patients were examined by immunofluorescence and transmission electron microscopy. The structure of eosinophil extracellular traps (EETs) and neutrophil extracellular traps (NETs) and stability against DNase were assessed in vitro. Platelet adhesion of EETs were also assessed.
Results:
Serum cf-nDNA and cf-mtDNA levels were significantly higher in AAV than in healthy controls, with the highest levels in EGPA; however, serum DNase activities were comparable among all groups. cf-nDNA and cf-mtDNA decreased after treatment and were associated with disease activity only in EGPA. Blood eosinophil count and plasma D-dimer levels were significantly correlated with cf-nDNA in EGPA and cf-mtDNA. EGPA tissue samples showed lytic eosinophils and EETs in small-vessel thrombi. The structure of EETs showed bolder net-like chromatin threads in vitro and EETs showed greater stability against DNase than NETs. EETs provided a scaffold for platelet adhesion.
Conclusion:
cfDNA was increased in EGPA, associated with disease activity. The presence of DNase-resistant EETs in small-vessel thrombi might contribute to higher concentration of cfDNA and the occurrence of immunothrombosis in EGPA.
Insights
Cell-free DNA (cfDNA) levels are elevated in antineutrophil cytoplasmic antibody-associated vasculitis (AAV), particularly in EGPA. Increased cfDNA in EGPA correlates with disease activity and may stem from DNase-resistant eosinophil extracellular traps (EETs).
Area of Science:
- Immunology
- Molecular Biology
- Pathology
Background:
- Cell-free DNA (cfDNA), originating from nuclear or mitochondrial DNA, is released into circulation upon cell damage.
- While cfDNA is linked to various diseases, its role in antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is not well understood.
- This study investigates the clinical significance of cfDNA in patients with AAV.
Purpose of the Study:
- To evaluate the clinical significance of cfDNA in antineutrophil cytoplasmic antibody-associated vasculitis (AAV).
- To compare cfDNA levels in different subtypes of AAV and healthy controls.
- To assess the relationship between cfDNA levels, disease activity, and treatment response in AAV.
Main Methods:
- Quantitative PCR was used to measure serum cf-nuclear DNA (cf-nDNA) and cf-mitochondrial DNA (cf-mtDNA) in 35 AAV patients (including EGPA, MPA, GPA) before and after immunosuppressive therapy.
- Immunofluorescence and transmission electron microscopy were employed to examine tissue samples from EGPA patients.
- In vitro studies assessed the structure and DNase stability of eosinophil extracellular traps (EETs) and neutrophil extracellular traps (NETs), as well as EETs' role in platelet adhesion.
Main Results:
- Serum cf-nDNA and cf-mtDNA levels were elevated in AAV patients compared to healthy controls, with the highest concentrations observed in EGPA.
- cfDNA levels decreased post-treatment and correlated with disease activity specifically in EGPA.
- EGPA tissues revealed lytic eosinophils and EETs within small-vessel thrombi; in vitro, EETs demonstrated greater DNase resistance than NETs and facilitated platelet adhesion.
Conclusions:
- Elevated cfDNA, particularly cf-nDNA and cf-mtDNA, is associated with AAV, most notably in EGPA.
- The presence of DNase-resistant EETs in thrombi may contribute to increased cfDNA concentrations and immunothrombosis in EGPA.
- cfDNA serves as a potential biomarker for disease activity in EGPA.
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