MDA5 multimerization on LINE RNA drives pathogenic extracellular immune complexes in autoimmunity
T. HsuiD, Xi Wang, Jiayan ZhangiD, Yukari Isayama, Viktoria E. M. Schindler, Eveline Van GompeliD, Hadil Maadadi, Celia Torres-AranciviaiD, Akiko Shimazaki-Takahashi, Nina KuriharaiD, Mariko Kondo, Marina GalesiciD, Antonella NotarnicolaiD, Sharmin SultanaiD, A. Maeda, Ruth Ann VleugelsiD, Paul F. DellaripaiD, Takashi YamashitaiD, Yukiko ItoiD, Kentaro AwajiiD, H. Kotani, Kazuki Mitsuru MatsudaiD, Begum HoruluogluiD, Caroline GrönwalliD, Vijay JoshuaiD, , Sun HuriD
Abstract
Autoantibodies are hallmarks of many autoimmune diseases, but their potential pathogenic roles, particularly for those targeting intracellular proteins, remain unclear. Anti-MDA5-positive dermatomyositis (anti-MDA5 DM) is characterized by autoantibodies against the intracellular protein MDA51,2, a conserved innate immune receptor that recognizes viral dsRNA by forming filaments3. Here, using four patient-derived monoclonal autoantibodies (mAbs), we reconstitute and define the molecular architecture, biogenesis, and immunological activity of pathogenic MDA5 immune complexes. Our cryo-EM analysis revealed that these mAbs bind dsRNA-scaffolded MDA5 filaments in at least two distinct binding modes, each exhibiting striking epitope convergence using germline-encoded residues. Extracellular immune complexes formed between mAbs and filamentous, but not monomeric, MDA5 potently activate multiple innate immune pathways, with the magnitude of activation determined by antibody binding mode and immune complex stoichiometry. Antibody bivalency further crosslinks MDA5 filaments into higher-order aggregates with heightened immunostimulatory activity, demonstrating an active role of autoantibodies in shaping immune complex architecture. Analysis of patient plasma reveals elevated levels of extracellular MDA5 filaments and identifies LINE retroelement-derived dsRNA as a structural scaffold. Notably, MDA5 immune complexes induce endogenous LINE dsRNA expression, likely promoting additional MDA5 filament formation and extracellular release through inflammatory cell death. These data thus support a self-amplifying inflammatory cycle as a pathogenic mechanism for anti-MDA5 DM. Collectively, our study defines a broadly applicable architectural principle, in which higher-order organization and binding modes of autoantibodies--beyond antibody affinity or nucleic acid presence alone--govern innate immune activation.
02 /Specification & Classification
Click any tag to browse matching papers- Citations
- 0
- Organism
- Assay
- Research Focus
- Approach
In-depth summary
Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (31 Jul 2026) · CC BY 4.0
Patient-derived anti-MDA5 antibodies bind MDA5 filaments in two germline-encoded modes; only filament immune complexes activate innate immunity, with LINE-derived dsRNA scaffolding circulating filaments in patient plasma.
Whether autoantibodies against intracellular proteins such as MDA5 are pathogenic, and which immune-stimulatory entity drives anti-MDA5 dermatomyositis, has been debated. This work rebuilds complexes from purified MDA5, RNA and antibody, showing that complex architecture, not affinity or nucleic acid presence alone, governs activation.
Key results
ShownFour patient mAbs fall into two classes. Class I binds monomeric and filamentous MDA5 equally; class II prefers filaments. Within each class the two mAbs converge on one epitope through germline-encoded residues.
Class I Kd = 2-8 nM for both forms; class II apparent Kd ~10-20 nM (filament) vs ~200-400 nM (monomer), ~8 to 38-fold. Class I epitope on Hel2i residues 670–690 (Lys685). Cryo-EM maps 2.09 to 3.67 Å; ELISA from 2 technical replicates. · Patient-derived mAbs bind MDA5 filaments in two…; Cryo-EM data processing and structure refinement; Figure legends
ShownOnly filamentous, not monomeric, MDA5 immune complexes drive type I/II interferon signaling in macrophages. FcγR blockade (CD64 strongest) reduced induction; TLR3/TRIF and MAVS loss suppressed it. Potency tracked binding mode, stoichiometry and class I filament bridging.
RNA-seq (2 biological repeats): NP6 + filament ICs induced IFN genes; monomer ICs, dsRNA-only and isotype controls were minimal. At MDA5:dsRNA 0.83:1 NP6 outperformed NP8; at 8.3:1 NP8 outperformed NP6. NP6 titration was bell-shaped; F01 beat F12. · MDA5 filament immune complex activates type I…; Anti-MDA5 mAbs bridge MDA5 filaments into…
ShownFilament ICs also trigger inflammatory cell death: ASC specks and NLRP3-dependent IL-1β release in THP-1 macrophages, and NETosis in healthy-donor neutrophils. Dropping MDA5, RNA or antibody abolished NETosis.
Basal NET frequency varied among donors (<1–8%); filament ICs with NP6 and NP8 robustly induced NETosis, monomeric ICs had no measurable effect. Neutrophils from three healthy donors, 2 biological repeats; ASC specks rose only with filament ICs. · MDA5 filament immune complexes activate…; Figure legends
SuggestedA TRIM65-based sandwich ELISA detects circulating antibody-bound MDA5 filaments. Levels are elevated in active anti-MDA5 DM, attenuated by glucocorticoids, and follow disease course in three longitudinal patients, in a small cohort.
TRIM65 ELISA detects filaments with 60-80-fold higher sensitivity than monomers. Cohort: 4 HCs, 19 DM (16 anti-MDA5-positive). Active no glucocorticoid n = 4, active on glucocorticoid n = 4, inactive n = 8. ICs fell with remission in P12 and P15 despite stable titers; rose in P18 despite falling titers. · MDA5 filament ICs are enriched in anti-MDA5 DM…; Figure legends
SuggestedLINE RNAs, mainly L1HS, are enriched in plasma and TRIM65-purified fractions of active anti-MDA5 DM and appear to be sense:antisense duplexes with A-to-I editing. They decline with remission and are induced by filament ICs in THP-1 cells.
L1HS sense-to-antisense ratio ~1.1 in plasma vs ~5.2 in PBMCs (P15, P12); approximately 2 edits per 100 bp. SINEs and ERVs not upregulated. Anti-MDA5-negative DM (P11, P13) lacked the enrichment. RNA-seq had 2 technical repeats. · LINE-derived RNAs are enriched in anti-MDA5 DM…; LINE-derived RNAs form dsRNA in plasma; LINE-derived RNAs are induced by immune…
Methods, data and reproducibility
Cryo-EM and biochemical characterization of four patient-derived mAbs on dsRNA-scaffolded MDA5 filaments, with reconstituted immune complexes tested in macrophages and neutrophils. Patient plasma was assayed by TRIM65 ELISA and spike-in-normalized stranded RNA-seq of plasma, TRIM65 pull-downs and PBMCs.
- Patient-derived anti-MDA5 mAbs: NP6 and NP8 from one Japanese patient (RP-ILD); F01 and F12 from Swedish anti-MDA5-positive patients · human · n = 4 mAbs
- Cryo-EM structures of MDA5ΔN filaments on dsRNA bound to scFv or Fab of the four mAbs · human mAbs; MDA5 species not stated · PDB: 21SJ, 22HM, 28BD, 28BE; EMD-67964, EMD-68271, EMD-81614, EMD-81615, EMD-68272, EMD68273
- RNA-seq of THP-1-derived macrophages stimulated with MDA5 ICs · human · n = 2 biological repeats · GSE315387
- Monocyte-derived macrophages and neutrophils from healthy donors stimulated with ICs · human · n = three healthy donors
- Brigham and Women's dermatomyositis plasma cohort for TRIM65 and direct ELISA · human · n = 4 HCs, 19 DM (16 anti-MDA5-positive)
- Stranded RNA-seq of plasma, TRIM65-purified plasma RNA and PBMCs from HCs, anti-MDA5 DM (P12, P15) and anti-MDA5-negative DM (P11, P13) · human · n = 2 technical repeats (not for TRIM65 RIP-seq) · GSE315387
- RefSeq viral genome reference for virome screening of patient RNA-seq · virus · n = n = 18,670
- Methods & tools
- cryoSPARC v4.7.1, ModelAngelo, COOT, PHENIX, UCSF ChimeraX, STAR v2.7.9a, DESeq2, TEtranscripts v2.2.4, EnrichR (Reactome 2024), Repbase, MAFFT, Trimmomatic v0.39
- Software & environment
- cryoSPARC v4.7.1; STAR v2.7.9a; Samtools v1.21; Trimmomatic v0.39; TEtranscripts v2.2.4; EnrichR with Reactome 2024; GRCh38.p14; Gencode v43. No container reported.
- Reproducibility gaps
- Deposited data are given as bare accessions (PDB, EMDB, GEO GSE315387; see methods.data), not URLs. Compute resources and container are not reported; access terms for patient sequencing data and antibody sequences are not stated.
Limitations (5)
- The initiating source of dsRNA that starts the inflammatory cycle remains uncertain; viral RNA was not enriched in patient plasma. · stated by the authors
- LINE induction is not a universal consequence of inflammation. It was absent in anti-MDA5-negative DM and SLE, and what distinguishes anti-MDA5 DM remains open. · stated by the authors
- Hel2i-loop flexibility limited class II resolution, so higher-resolution NP8 and F12 structures used a loop-shortened MDA5 construct with the G495R filament-stabilizing mutation. · stated by the authors
- The patient cohort is small (4 HCs, 19 DM) and treatment-heterogeneous (IVIG, mycophenolate, glucocorticoids). Plasma RNA-seq rests on few patients (P12, P15 vs P11, P13), so links to disease activity are preliminary. · noted in this summary
- Pathogenic activity is shown only with reconstituted recombinant complexes in THP-1 cells and healthy-donor primary cells in vitro. The self-amplifying loop is inferred from separate experiments, not tested in vivo. · noted in this summary
Who should read:Read this if you study autoantibody-nucleic acid immune complexes, MDA5 or RIG-I-like receptor biology, or LINE retroelement RNA as an extracellular innate immune ligand in autoimmunity.
Reproducibility ledger
Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.
Sign in to file a reproducibility report.
Related papers
- A dynamic molecular glue targets a plastic MDA5 filament interface to stimulate innate immunity
- Molecular basis of autoimmune disease protection by MDA5 variants
Rahul Singh, Joe D. Joiner, … Yorgo Modis·Cell ReportsPeer-reviewed·May 30, 2025
- Anti-MDA5 monoclonal antibodies from patients with dermatomyositis - B cell characteristics and differential targeting of the helicase domains
Eveline Van Gompel, Marina Galesic, … Vijay Joshua·Journal of AutoimmunityPeer-reviewed·Feb 19, 2026
- Establishing the molecular basis for MDA5 mutation-linked autoimmunity
Ling Xu, Harim Jang, … Anna Marie Pyle·Nucleic Acids ResearchPeer-reviewed·Sep 22, 2026
- Heteromeric LGP2-MDA5 receptor complexes drive antiviral signaling
Gina Buchel, Ling Xu, … Anna Marie Pyle·bioRxiv·Oct 19, 2026
- A Small Subset of Cytosolic dsRNAs Must Be Edited by ADAR1 to Evade MDA5-Mediated Autoimmunity
Tao Sun, Qin Li, … Jin Billy Li·bioRxiv·Aug 29, 2022