Background/Purpose: Genetic studies of new-onset juvenile dermatomyositis (JDM) exhibit elevation of Type 1 interferons (IFN 1) IFNα and IFNβ in blood, skin, and muscle. To understand the relationship of IFN 1 to muscle dysfunction we used an in vitro 3D biomimetic construct derived from healthy pediatric muscle (myobundles). IFN 1 reduces myobundle contractile force, upregulates MHC class 1 and induces donor-specific expression of myositis-specific antigens. Janus kinase inhibitors (JAKi) have therapeutic effect in Type 1 interferonopathies. In this study we evaluated gene expression of myobundles exposed to IFNα or β as well as after cotreatment with IFNβ and JAKi, tofacitinib or baricitinib.

Methods: Myoblasts isolated from 3 healthy pediatric donors were cultured and used to create donor-specific myobundles, exposed to 0 (control), 5, 10 or 20 ng/mL IFNα or β. In a separate protocol myobundles were exposed to 0 (control) or 20 ng/mL IFNβ for a total of 10 days then treated with tofacitinib 1 µM or baricitinib 500 nM from days 3-10 of IFNβ exposure. Myobundle mRNA was isolated for sequencing. Quality profiling and alignment of mRNA was performed using the fastp toolkit and splice aware STAR RNA-seq alignment tool, respectively. Differential expression was performed using the DESeq2 Bioconductor package. Independent filtering was used prior to calculating adjusted p-values, and moderated log2 fold-changes were derived using the ashr package. Gene set enrichment analysis was performed using hallmark pathways associated with altered gene expression for each comparison performed.

Results: Eighty-three myobundles from 3 healthy pediatric donors were analyzed. PCA analysis indicates distinct clusters of similar gene expression across IFNα-exposed and IFNβ-exposed myobundles vs. untreated controls, respectively (Figure 1). IFNβ-exposed myobundles treated with JAKi have gene profiles more similar to controls. The top 5 most overexpressed and downregulated genes after exposure to 20 ng/mL IFNα or IFNβ vs. control are listed in Table 1 and included interferon response genes and proinflammatory genes. Gene Set Enrichment Analysis showed that IFNα induced enrichment of 29/50 pathways (58%) with IFNβ enrichment of 33/50 pathways (66%). Key enriched pathways included IFNα and γ response genes and inflammatory response genes. IFNα downregulated oxidative phosphorylation genes while IFNβ decreased expression of myogenesis genes (Figure 2). Treatment with IFNβ and JAKi had significantly less pathway enrichment (baricitinib with 13/50 and tofacitinib 16/50 enriched) with key enriched pathways involving skeletal muscle myogenesis.

Conclusion: IFNα and IFNβ have distinct effects on pediatric skeletal muscle gene expression, with upregulation of IFN and inflammatory response genes and downregulation of oxidative phosphorylation and myogenesis genes. Genetic profiles are significantly altered by JAKi, baricitinib and tofacitinib. Results demonstrate the potential of bioengineered pediatric skeletal muscle to advance our knowledge of JDM pathogenesis and provide a platform for investigating promising therapeutics.

Figure 1. PCA plot of all myobundle samples. Green arrows show shift after IFNα; black arrows show greater shift in PC1 after IFNβ.

Table 1. Most overexpressed and downregulated genes after IFN 1 exposure in pediatric skeletal myobundles. lncRNA = long non-coding RNA; IRG = interferon response gene

Figure 2. GSEA pathways after IFN I exposure

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/effect-of-type-1-interferons-and-jak-inhibitors-on-gene-expression-in-bioengineered-pediatric-skeletal-muscle/