Clarifying the pathogenesis mechanism of eosinophilic granulomatosis with polyangiitis (Teppei Hashimoto, Lecturer, Department of Diabetes and Immunology)

A paper by Lecturer Teppei Hashimoto Department of Diabetes Endocrinology and Clinical Immunology and his colleagues was published in the international academic journal "Frontiers in Immunology" (January 12, 2022).

This study clarified the pathogenesis mechanism of eosinophilic granulomatosis with polyangiitis, an autoimmune systemic vasculitis.

For details about the research, please see below.

Increased circulating cell-free DNA in eosinophilic granulomatosis with polyangiitis: implications for eosinophil extracellular traps and immunothrombosis

Teppei Hashimoto※1, Shigeharu Ueki※2, Yosuke Kamide※3, Yui Miyabe※2, Mineyo Fukuchi※2, Yuichi Yokoyama※1, Tetsuya Furukawa※1, Naoto Azuma※1, Nobuyuki Oka※4, Hiroki Takeuchi※5, Kyoko Kanno※6, Akemi Ishida-Yamamoto※6, Masami Taniguchi※3, Akira Hashiramoto※7, Kiyoshi Matsui※1

*1 Department of Department of Diabetes Endocrinology and Clinical Immunology Hyogo Medical University
*2 Department of General Medicine and Laboratory Diagnostics, Graduate Graduate School of Medicine, Akita University
*3 Sagamihara Hospital, National Hospital Organization
*4 Kyoto Konoe Rehabilitation Hospital
*5 Nankyoto Hospital, National Hospital Organization, Independent Administrative Institution
*6 Department Department of Dermatology Asahikawa Medical University
*7 Department of Clinical Diagnosis and Laboratory Medicine, Graduate School of Health Sciences, Kobe University

Eosinophilic granulomatosis with polyangiitis (EGPA) is an autoimmune systemic vasculitis characterized by the appearance of neutrophil cytoplasmic antibodies (ANCA). Eosinophils play an important role in the pathogenesis, but the underlying mechanism remains unclear.

We focused on cell death (EETosis) caused by eosinophil extracellular traps (EETs), which are important in allergic inflammation, and the DNA fragments released from these cells (cell-free DNA: cfDNA), and investigated these using patient serum and pathological tissues. Results revealed that high concentrations of cfDNA were present in peripheral blood of patients with EGPA during the active phase and correlated with thrombosis markers. Pathological tissue revealed that intravascular thrombi were filled with eosinophils undergoing EETosis and released DNA from their nuclei. Furthermore, we found that EETosis is poorly soluble in DNase, and platelets aggregate using it as a scaffold to promote thrombus formation.

This study demonstrated that activated eosinophils in the blood vessels of EGPA patients cause EETosis, which leads to a condition known as Department of Immunology thrombosis, and that cfDNA may be a useful biomarker for this condition.

ANCA-associated vasculitis is a necrotizing vasculitis affecting small blood vessels and is classified as EGPA, granulomatosis with polyangiitis (GPA), and microscopic polyangiitis (MPA). Abnormalities in neutrophil extracellular traps (NETs) have been reported as a mechanism for ANCA production. Activated neutrophils form NETs, and myeloperoxidase (MPO) released extracellularly along with DNA acts as an autoantigen, inducing MPO-ANCA production, which in turn induces NETs, creating a vicious cycle.

On the other hand, unlike GPA and MPA, EGPA is characterized by the fact that not only ANCA and neutrophils but also eosinophils play an important role in the pathogenesis. Eosinophils are also activated by stimuli such as IL-5 and LPS, and upon degranulation release DNA outside the cell to form EETs. cfDNA is used as a biomarker for diseases associated with NETs and is predicted to be an important marker for EETs.

We quantitatively measured cfDNA extracted from serum of patients with EGPA, GPA, and MPA before and after treatment using real-time PCR and analyzed its correlation with disease parameters. Blood cfDNA concentrations were significantly higher in EGPA than in GPA or MPA, reflecting disease activity, and correlations with eosinophils and D-Dimer were also observed. Furthermore, pathological tissue obtained from skin and nerve biopsies of EGPA patients were examined for EETs/EETosis in thrombi using HE staining, immunofluorescence staining, and electron microscopy. Immunostaining for the vascular endothelial marker CD31 and the EETs markers citrullinated histone and DNA, and comparing the results with HE staining, demonstrated the presence of EETs/EETosis in blood vessels.

Next, we stimulated eosinophils and neutrophils extracted from peripheral blood with PMA to induce EETs/NETs. After DNase treatment, we compared the rate of DNA degradation released from the nuclei. EETs were more resistant to DNase than NETs, and DNA degradation took longer. To further assess whether EETs promote thrombus formation, we incubated plasma containing fluorescently labeled platelets with EETosis cells and quantified the number of adherent platelets. Scanning electron microscopy revealed that numerous platelets adhered to EETosis cells, and the number of adherent platelets decreased when EETs were removed by DNase.

The results of this study revealed that in EGPA, activated eosinophils in blood vessels cause EETosis, promoting thrombus formation, and that cfDNA is a useful biomarker for this.

2019 Faculty Research Grants

This research was conducted in collaboration with the Department of General Medicine and Laboratory Diagnostics, Graduate Graduate School of Medicine Akita University, and National Sagamihara Hospital.

We demonstrated that eosinophil activation, cell death, and associated thrombus formation are important in EGPA, suggesting that treatments targeting eosinophils are important in this disease. Future challenges include developing new therapies that inhibit eosinophil activation, and suppressing this intractable pathology.