Elevated ISG15 reinforces RIG-I/MDA5-linked macrophage-microglia crosstalk during blood-brain barrier disruption in neuromyelitis optica spectrum disorder.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is an AQP4-IgG-mediated astrocytopathy characterized by blood-brain barrier (BBB) injury and myeloid inflammation. We investigated whether interferon-stimulated gene 15 (ISG15) contributes to myeloid activation through RIG-I-like receptor (RLR) signaling. METHODS: Single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) from patients with NMOSD was combined with validation in monocytes, serum, and cerebrospinal fluid. THP-1-derived macrophages and HMC3/BV2 microglia underwent LPS and/or IFN-β stimulation and ISG15 knockdown or overexpression. To assess macrophage-derived ISG15 effects on microglial polarization, ISG15-manipulated macrophages were non-contact cocultured with microglia in Transwell inserts, and microglial M1/M2 markers were measured. Western blotting quantified free and conjugated ISG15. ISG15-RIG-I association were examined by co-IP. An AQP4-IgG/human complement passive-transfer mouse model provided in vivo validation. RESULTS: NMOSD PBMCs showed monocyte expansion and an ISG15-high interferon/RLR signature, with increased ISG15 in CD14+ monocytes, serum, and cerebrospinal fluid. In macrophages, LPS plus IFN-β increased inflammatory cytokines, migration, and both free/conjugated ISG15. ISG15 overexpression or knockdown bidirectionally altered macrophage and microglial phenotypes; the Transwell coculture confirmed that macrophage ISG15 status directly modulated microglial M1/M2 polarization. ISG15 modulated RIG-I/MDA5/LGP2 expression, with RIG-I overexpression partially rescuing the RLR program after knockdown. Co-IP confirmed physical association between ISG15 and RIG-I. In vivo, BBB disruption coincided with ISG15-high infiltrating and resident myeloid cells and RLR upregulation. CONCLUSION: Elevated ISG15 following BBB injury reinforces RIG-I/MDA5/LGP2 signaling and drives macrophage-microglia inflammatory crosstalk in NMOSD, establishing the ISG15-RLR axis as a key amplification pathway that bridges peripheral and central myeloid responses.