Recapitulation of pro-inflammatory signature of monocytes with ACVR1A mutation using FOP patient-derived iPSCs

Abstract

Background Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by progressive heterotopic ossification (HO) in soft tissues due to a heterozygous mutation of the ACVR1A gene (FOP-ACVR1A), which erroneously transduces the BMP signal by Activin-A. Although inflammation is known to trigger HO in FOP, the role of FOP-ACVR1A on inflammatory cells remains to be elucidated. Results We generated immortalized monocytic cell lines from FOP-iPSCs (FOP-ML) and mutation rescued iPSCs (resFOP-ML). Cell morphology was evaluated during the monocyte induction and after immortalization. Fluorescence-activated cell sorting (FACS) was performed to evaluate the cell surface markers CD14 and CD16 on MLs. MLs were stimulated with lipopolysaccharide or Activin-A and the gene expression was evaluated by quantitative PCR and microarray analysis. Histological analysis was performed for HO tissue obtained from wild type mice and FOP-ACVR1A mice which conditionally express human mutant ACVR1A gene by doxycycline administration. Without any stimulation, FOP-ML showed the pro-inflammatory signature of CD16+ monocytes with an upregulation of INHBA gene, and treatment of resFOP-ML with Activin-A induced an expression profile mimicking that of FOP-ML at baseline. Treatment of FOP-ML with Activin-A further induced the inflammatory profile with an up-regulation of inflammation-associated genes, of which some, but not all, of which were suppressed by corticosteroid. Experiments using an inhibitor for TGFβ or BMP signal demonstrated that Activin-A-induced genes such as CD16 and CCL7, were regulated by both signals, indicating Activin-A transduced dual signals in FOP-ML. A comparison with resFOP-ML identified several down-regulated genes in FOP-ML including LYVE-1, which is known to suppress matrix-formation in vivo. The down-regulation of LYVE-1 in HO tissues was confirmed in FOP model mice, verifying the significance of the in vitro experiments. Conclusion These results indicate that FOP-ML faithfully recapitulated the phenotype of primary monocytes of FOP and the combination with resFOP-ML is a useful tool to investigate molecular events at the initial inflammation stage of HO in FOP.

Intersections of Fibrodysplasia Ossificans Progressiva and Traumatic Heterotopic Ossification

“…When monocytes collected from FOP patients were stimulated with lipopolysaccharide (LPS), they showed the prolonged activation of NF-κB, suggesting its role in FOP inflammation [37]. These and other findings have shown that ACVR1 activity causes a pro-inflammatory state through increased NF-κB [183]. Additionally, transforming growth factor beta (TGF-β), a cytokine released by monocytes and macrophages, is increased in FOP patients and has been shown to attenuate HO formation in FOP mouse models when systemically suppressed [37,118].…”

Novel Therapeutic Targets for Fibrodysplasia Ossificans Progressiva: Emerging Strategies and Future Directions

“…In order to produce healthy bone-forming cells that can replace the abnormally formed bone, researchers are investigating the use of iPSCs in FOP. This strategy shows potential for regenerating healthy tissue and functionality [45,46]. A number of transcription factors, including Oct4, Sox2, Klf4, and c-Myc, can be added to somatic cells to produce iPSCs.…”

“…Researchers are looking into several immunotherapeutic strategies that can specifically target the cells responsible for aberrant bone growth, such as monoclonal antibodies and immune checkpoint inhibitors. These treatments seek to prevent or lessen HO in FOP by regulating the immunological response [9,46]. Monoclonal antibodies are proteins that can attach to particular antigens on the surface of cells and cause the immune system to start destroying those cells.…”

Human iPSCs as Model Systems for BMP-Related Rare Diseases

“…Notable differences were observed in FOP iPSC-derived M1 macrophages, which exhibited higher expression levels of IL6, IL1a, RANTES, and Activin-A, among others. Maekawa et al pursued a different approach by differentiating isogenic iPSC lines into the monocytic lineage and subsequently immortalizing the cells [128,129]. FOP immortalized monocytes (FOP-ML) displayed elevated levels of phosphorylated SMAD5, both at basal levels and upon stimulation with recombinant Activin-A, unlike the control cells.…”