Screening and characterization of myositis-related autoantibodies in COVID-19 patients

Abstract

An efficient host immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) appears to be crucial for controlling and resolving this viral infection. However, many studies have reported autoimmune characteristics in severe COVID-19 patients. Moreover, clinical observations have revealed that COVID-19-associated acute distress respiratory syndrome shares many features in common with inflammatory myopathy including interstitial lung disease (ILD), most particularly rapidly progressive (RP)-ILD. This study explored this phenomenon by seeking to identify and characterize myositis-specific and related autoantibodies in 25 COVID-19 patients with mild or severe symptoms. Line blot analysis with the EUROLINE Myopathies Ag kit identified 9 (36%) patients with COVID-19 with one or more autoantibodies against several myositis-related antigens (Jo-1, Ku, Mi-2β, PL-7, PL-12, PM-Scl 75, PM-Scl 100, Ro-52, and SRP); no anti-MDA5 antibodies were detected. As the presence of antibodies identified by line blots was unrelated to disease severity, we further characterized the autoantibodies by radioimmunoassay, in which [³⁵ S]methionine-labeled K562 cellular antigens were precipitated and visualized by gel electrophoresis. This result was confirmed by an immunoprecipitation assay and immunoblotting; 2 patients exhibited anti-Ku70 and anti-Ku80 antibodies. Our data suggest that it is necessary to use more than one method to characterize and evaluate autoantibodies in people recovered from COVID-19, in order to avoid misinterpreting those autoantibodies as diagnostic markers for autoimmune diseases.

FIGURE 1

Immunoprecipitation analysis of antigens recognized by autoantibodies purified from sera of either interstitial lung disease patients (ILD) or COVID‐19 patients (COVID). [³⁵S]Methionine‐labeled antigens were extracted from K562 cells after overnight incubation with [³⁵S]methionine‐containing medium. Arrowheads and parentheses indicate positions of autoantibody‐recognized antigens (red solid arrowheads and red parentheses: recognized by autoantibodies of sera from ILD patients; red empty arrowheads: recognized by autoantibodies of sera from COVID‐19 patients). Molecular weight is indicated on the left‐hand side of each figure. HC, healthy control. Left (a), exposed for 7 days. Right (a), exposed for 14 days. (b) exposed for 7 days. Dashed line: removal of the lane due to repeated data of lane 3 (patient 6).

FIGURE 2

Immunoprecipitation followed by Western blot was performed to visualize specific proteins recognized by autoantibodies from patient sera. (a) Immunoprecipitation of Ku70 protein. (b) Immunoprecipitation of Ku80 protein. (c) Immunoprecipitation of PL‐7 protein. Molecular weight is indicated on the left‐hand side of each figure.