A role for primary cilium in muscular interferonopathies – MYOCILINF-2023

c.1. WP 1: Dissection of the muscular molecular signature in IBM
Task 1. Characterization of the whole molecular signature in muscle tissue: bulk RNAseq
Task 2. Cellular scale and spatial characterization of molecular signatures in IBM muscle: sn- and spatial transcriptomics
c.2. WP 2: Effects of IFN? on primary cilium
Task 1 Signal transduction by IFN? receptors : to generate both stable immortalized IBM and control primary cilium reporter cell lines through overexpression of Arl13b-GFP proteins
Task 2 IFN?-induced primary cilium defects : The human mpc cells will be exposed to Cytochalasin D, Ciliborevin D, Chloral hydrate and SAG, then analyzed by western-blotting (IFT proteins) and/or RT-qPCR (Ptch1, Smo, Gli3 a minima). For imaging analysis, regarding the size of intra-flagellar trains in primary cilium, we will preferentially use high-resolution STimulated Emission Depletion (STED) nanoscopy to detect (at least) both IFT20 (involved in both transport from endoplasmic reticulum to primary cilium and, belonging to IFT-B complex involved in anterograde transport) and IFT140 proteins (belonging to IFT-A complex involved in retrograde transport).
c.3. WP 3: Experimental analysis of the myosuppressive effects of IFN? in mouse models
Task 1. Evaluation of the effects of protracted exposure to IFN? on muscle structure and function We will evaluate the myological status in IFN? mice and controls (pump with vehicle) mice and C57BL/6*DBA/2 CTL at 1, 3 and 6 months (12 animals/group; total: n=96) using the following approaches: Muscle function analysis, myopathological study, myogenic progenitor cells (mpc) behavior, primary cilium evaluation
Task 2. Evaluating the effects muscle injuries IFN? on muscle structure and function: Effects of long-term exposure to IFN? on muscle repair
c.4. WP 4: Evaluation of primary cilium in IBM muscles and controls
Task 1. Evaluating the signaling pathways in primary cilium: RT-qPCR analysis
Task 2. Evaluating the role of ectosomes: We will use human mpc cultures to promote primary cilium elongation (through serum deprivation). These mpc cultures will be exposed to pharmacological compounds including JAK/STAT inhibitor and the both activator (SAG) and inhibitor (Ciliobrevin D) of hedgehog signaling pathway. The analysis will be performed by immunocytochemistry and western-blotting analysis.
Task 3. Evaluating the primary cilium defects : We will use electronic microscopy to identify major structural defects in primary cilium. Concomitantly, we will use high-resolution 3D fluorescence microscopy (by structured illumination microscopy [SIM]) to evaluate the ring-shaped organization of the basal body (BBSome including BBS9 and BBS12) ring-shaped organization, the structural organization of transition zone (through the study at least of CEP290) and the presence and composition of IFT trains (IFT-B and IFT-A complexes members).

Through WP1 of the MYOCILINF-2023 project, we identified that :
1) human PAX7+ MuSCs harbor primary cilium aligned with the myofiber, potentially positioning the cilium to more effectively sense signals distributed along the fiber surface;
2) human myogenic cells harbor primary cilium during the differentiation process, suggesting that this organelle may remain functional during early stages of myogenic differentiation prior to fusion;
3) Primary cilium undergoes elongation during MuSCs commitment to differentiation, indicating that primary cilium remodeling accompanies transitions between myogenic cell states and that ciliary elongation in committed myogenic cells. This suggests that primary cilium contributes to signaling events regulating human stem cell fate decisions during early stages of human myogenic differentiation;
4) Activation of Hedgehog signaling during myogenic differentiation of MuSCs is driven by INDIAN HEDGEHOG. These data were confirmed by GLI3 and GPR161 level and location analyses;
5) INDIAN HEDGEHOG levels increasing during myogenic differentiation and is the sole ligand involved in myogenic differentation contrary to SONIC HEDGEHOG and DESERT HEDGEHOG ligands;
6) early MuSC differentiation is associated with increased expression and secretion of the INDIAN HEDGEHOG ligand;
7) Transcriptomic and proteomic analyses revealed that GLI1, the main transcriptional activator and canonical target of Hedgehog signaling, is strongly correlated with expression of MYOG (a key marker of MuSC commitment to myogenic differentiation) and upregulated during myogenic differentiation;
8) Primary cilium depletion by siRNA ARL13b and IFT88 impairs myogenic differentiation and alters MuSC fate progression, indicating that the primary cilium is required for efficient progression of human MuSCs through the myogenic differentiation program and suggest that ciliogenesis contributes to the regulation of Hedgehog signaling during early stages of myogenic commitment.

Taken together these data will allow to propose a model of IFN?-induced PC shedding in human primary myoblasts. We are ongoing developping a skeletal muscle cell lines wich expressed fluorescent primary cilium to evaluate in live imaging the effects of IFN? in human muscle precursor cells. Further publication will include: 1/ the generation of a human muscle cell line with a fluorescent primary cilium; 2/ the optimization of U-ExM on human muscle samples and human biposies; 3/ the model of IFN?-induced PC shedding in human primary myoblasts.
Then, we evaluate the role of primary cilium in both IBM and controls muscles. Through WP4 of the MYOCILINF-2023 project, we already identified that :
14) Normal ageing is associated with PC legnth decrease in human primary myogenic precursor cells;
15) After 50-years old (and until 70-years old at least), the primary cilium length is conserved;
16) Primary cilium length defects are observed in human myogenic precursor cells from IBM patients
17) Presence of vesicular budding at the ciliary membrane is increased in human myogenic precursor cells from IBM patients;
18) Abnormal accumulation of IFT20 proteins is observed at the basal body of primary cilium in human myogenic precursor cells from IBM patients;
19) ARL13B, a primary cilium membrane specific protein, was found accumulated in muscle tissue sections of IBM patient;
20) In control patient sections, ARL13B abundance differed according to the fiber type: type I fibers displayed significantly higher ARL13B intensity than type II fibers, both in the sarcoplasm and at the sarcolemma level, suggesting a potential role for ARL13B in myofiber basal metabolism and muscle physiology.
21) ARL13B fluorescence intensity is markedly increased in IBM patient myofibers compared to controls, across both type I and type II fibers. Moreover, the fiber-type-specific distribution of ARL13B observed in myofibers from control patients was no longer present in IBM patient myofibers, suggesting a global dysregulation of ARL13B protein in IBM muscle.

Altogether, these results demonstrate a primary cilium defect in myoblasts from IBM patients in vitro, along with a dysregulation of the key ciliary component ARL13B in IBM patients myofibers, suggesting a role of the PC in the pathophysiological mechanisms of IBM and that IBM is a muscle ciliopathy.

Hou C, Periou B, Gervais M, Martin L, Berthier J, Baba-Amer Y, Souvannanorath S, Lechapt-Zalcman E, Malfatti E, Gherardi RK, Relaix F, Bencze M, Authier FJ. Interferon-? causes myogenic cell dysfunction and senescence in immune myopathies. Brain. 2025 Aug 1;148(8):2883-2898. doi: 10.1093/brain/awaf153. PMID: 40296760.

Inclusion body myositis (IBM) differs from other autoimmune myositis by its poor response to immunosuppressant therapies, and degenerative muscular features leading to marked disability. IBM is characterized by a strong IFN-gamma (IFN?) signature in muscle tissue, which is related to the CD8+ T-cells infiltrating muscles and producing large amounts of IFN?. The pathophysiology of IBM has been mainly regarded from the view of immune response, so that the mechanisms leading to muscle degenerative features remains largely unknown. We previously showed that (i) in IBM, muscle cells themselves display evidences for IFN? signaling; (ii) in vitro, IFN? impairs the proliferation and differentiation of myogenic cells and will induce their senescence; and (iii) in vivo, the combination of a muscular injury with IFN? administration is sufficient to reproduce some degenerative muscular features of IBM. In addition, we also showed that in IBM, IFN? signature strongly correlates with primary cilium signature. From these results, we hypothesized that IFN? could be the key factor of muscular degeneration in IBM and primary cilium disrupting and/or dysfunction could, at least in part, mediate the pathogenic effects of IFN? in IBM. Our project aims (i) to dissect at the cell and molecular levels the effects of IFN? in IBM patients by using single nucleus RNAseq and spatial transcriptomics; (ii) to analyze in vitro the effects of IFN? on primary cilium on human myogenic cells; (iii) to deepen the in vivo effects of IFN? on skeletal muscle biology with a special attention to primary cilium structure and function; and (iv) to validate in patients the experimental results dealing with the effects of IFN? on primary cilium. Evidencing that IBM is an acquired IFN?-driven ciliopathy represents a conceptual breakthrough in myology. Primary cilium has never been investigated in the context of muscular diseases. We believe that present project will pioneer the field of muscular ciliopathy.