Comparative analysis between different types of autoantibodies and their effects on NETs formation

Background and Aims Anti-neutrophil cytoplasmatic antibodies (ANCA) are found in serum of patients with autoimmune hepatitis, primary sclerosing cholangitis and primary biliary cirrhosis. ANCA levels were shown to be associated in liver cirrhosis with disease severity and risk of infections. Moreover, ANCAs were described to induce neutrophil extracellular traps (NETs) release and NETs were shown to promote autoantibodies formation. We aimed to study if different types of autoantibodies differentially affect NETs formation.

Method Neutrophils were isolated from healthy volunteers’ peripheral blood with Polymorphprep (Axis-shield, Oslo, Norway). Cells were challenged with vehicle or 12 µg/ml of perinuclear ANCA (pANCA) (anti-MPO antibody, Dako, Santa Clara, CA, USA), rabbit IgG isotype control (Dako), cytoplasmic (cANCA) (anti-PR3 antibody, Immunotools, Friesoythe, Germany), antinuclear antibodies (ANA) (anti-citHistone3 antibody, abcam, Cambridge, UK) in presence of medium, heat-inactivated E. coli (250 bacteria/cell) or phorbol 12-myristate 13-acetate (PMA) (100nM). After incubation for 2 hours at 37°C cells were fixed with paraformaldehyde and stained with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Carlsbad, CA, USA). Percentage of NETs formation was assessed with Olympus BX51 Fluorescence Microscope (Olympus, Shinjuku, Tokyo, Japan) at 600x total magnification and quantified as NETs-like structures divided by total number of neutrophils.

Results NETs formation was significantly induced by pANCA (Mean±SD = 35.1±18.3%, p=0.019), but not rabbit IgG isotype control (Mean±SD = 9.6±5.5%), cANCA (Mean±SD=5.4±3.2%) or ANA (Mean±SD=7±5.9%) in healthy donor derived unstimulated neutrophils compared to vehicle control. No significant influence of pANCA on NETs formation in response to E. coli or PMA was found.

Conclusion Our finding that pANCA, but not cANCA or ANA autoantibodies cause NETs formation emphasizes a crosstalk between adaptive immunity and deranged innate immune function as a feature of autoimmunity and therefore may improve understanding of the disease and provide basis for future targeted treatment to break NETs-ANCA vicious cycle.

 

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Artikel online veröffentlicht:
26. August 2022