Interleukin-17B is a new biomarker of human muscle regeneration in dystrophinopathies

Abstract

We aimed to identify novel biomarkers of muscle pathological changes via a large-scale histopathology-based multi-omics study of dystrophinopathies. We performed a comparative pathological analysis of 121 Duchenne muscular dystrophy (DMD) and 114 Becker muscular dystrophy (BMD) patients to determine muscle pathological similarities and differences between DMD and BMD that have not been systematically investigated. Customized bioinformatic analyses of bulk muscle RNA-sequencing data derived from 35 DMD patients, 39 BMD patients, and 21 controls were performed to identify gene signatures associated with pathological changes. Validation experiments, including single-nucleus RNA-sequencing, RNAscope in situ hybridization, and immunofluorescence staining, were performed in a subset of DMD and BMD patients, as well as 27 patients with other acquired and inherited myopathies. Systematic pathological analyses revealed that the percentages of necrotic, regenerating, and hypercontractive myofibers and the degree of muscle fibrosis were greater in DMD patients than in BMD patients. In both DMD and BMD patients, the percentages of necrotic, regenerating, and hypercontractive myofibers respectively increased in the early-stage and decreased in later disease stages, whereas muscle fibrosis progressively worsened with disease progression. Comparative transcriptomic analysis indicated that inflammatory responses were significantly activated in DMD patients compared to BMD patients, which was confirmed by immunohistochemistry analyses. Our customized bioinformatic analyses identified the gene set of MYH3, MYH8, IL17B, TNNT2, MYMK, and TDO2 as the most associated gene signature for muscle necrosis and regeneration. Muscle quantitative reverse transcription-polymerase chain reaction analyses confirmed significantly increased levels of IL17B and TNNT2 mRNA expression in both DMD and BMD patients compared to controls. Muscle IL17B mRNA expression was significantly correlated with histological muscle regeneration and negatively correlated with the age of patients with dystrophinopathy. Single-nucleus RNA-sequencing and RNAscope in situ hybridization demonstrated that IL17B mRNA was expressed in regenerating myofibers of patients with DMD and BMD, as well as in various acquired and inherited myopathies. Immunofluorescence staining further confirmed that interleukin-17B was expressed in regenerating myofibers of DMD and BMD patients. Our study provides evidence that interleukin-17B is a new biomarker of muscle regeneration in dystrophinopathies.