Background: Neurogenic heterotopic ossifications (NHOs) are incapacitating extra-skeletal bones that form in periarticular muscles after damage to the central nervous system such as spinal cord injuries (SCI). NHOs cause joint ankylosis, as well as vascular and nerve compression, with the only effective therapy being surgical resection. Using our mouse model of NHO, we previously identified an endogenous glucocorticoid switch driving NHO after SCI. Remarkably, mice with a tamoxifen-inducible post-natal deletion for the glucocorticoid receptor gene Nr3c1 in all cells (Nr3c1^flox/flox|Rosa26^CreERT2) did not develop NHO, and short-term administration of glucocorticoid receptor antagonists (mifepristone, relacorilant) significantly inhibited NHO development.

Method: NHO was induced in mice by combining a muscle injury via intramuscular injection of cardiotoxin, with a SCI between vertebrae T11-13. To identify genes involved in NHO development, bulk RNA sequencing was performed in C57BL/6 mice that underwent muscle injury via an intramuscular injection of cardiotoxin +/- SCI and +/- glucocorticoid receptor (GR) antagonist relacorilant treatment. In addition, mice with a conditional knock for the glucocorticoid receptor gene Nr3c1 in muscle satellite cells (Nr3c1^flox/flox|Pax7-CreERT2), mesenchymal fibro-adipogenic progenitors (Nr3c1^flox/flox|Pdgfra-MerCreMer) and myeloid cells (Nr3c1^flox/flox|Lyz2-Cre) were utilised.

Aims and Results: RNAseq analysis at 4 days post-surgery revealed that several pro-osteogenic and pro-inflammatory genes are induced by SCI in injured muscles and downregulated in response to relacorilant (Runx2, Creb3l1, Ddr2, Lrp8, Vdr, Ccn4, Fstl3, Thbs1, Spock2, Osm, IL1b). Likewise, many genes involved in antiviral and interferon-mediated responses were upregulated by SCI and downregulated by relacorilant (Ifnar1, Ifnar2, Rigi, Dhx58, Ifih, Aim2, Sting1, Mx1, Irf7). Conditional deletion of the Nr3c1 gene in muscle Pax7+ satellite cells had no significant impact on NHO development after SCI. In contrast, Nr3c1 deletion in either mesenchymal or myeloid cells altered NHO development in response to SCI.

Conclusions:  

SCI causes abnormal activation of pathways involved in osteogenic, inflammatory, antiviral and interferon-mediated responses, which are reduced following GR inhibition. Endogenous glucocorticoids drive NHO development after SCI via GR expressed by myeloid and mesenchymal cells. Overall, this study may reveal additional therapeutic targets to prevent NHO development in victims of SCI.