Neutrophil-microglia interaction drives motor dysfunction in a neuromyelitis optica model induced by subarachnoid AQP4-IgG
Fangfang Qi, Vanda A. Lennon, Shunyi Zhao, Yong Guo, Husheng Ding, Caiyun Liu, Whitney M. Bartley, Tingjun Chen, Claudia F. Lucchinetti, Long-Jun Wu
Fangfang Qi, Vanda A. Lennon, Shunyi Zhao, Yong Guo, Husheng Ding, Caiyun Liu, Whitney M. Bartley, Tingjun Chen, Claudia F. Lucchinetti, Long-Jun Wu
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Research Article Autoimmunity Neuroscience

Neutrophil-microglia interaction drives motor dysfunction in a neuromyelitis optica model induced by subarachnoid AQP4-IgG

Abstract

Neutrophils and neutrophil extracellular traps (NETs) contribute to early neuromyelitis optica (NMO) histopathology initiated by IgG targeting astrocytic aquaporin-4 (AQP4) water channels. Yet, the mechanisms underlying neutrophil recruitment and their pathogenic roles in disease progression remain unclear. To investigate molecular-cellular events preceding classical complement cascade activation in a mouse NMO model, we continuously infused, via spinal subarachnoid route, a non-complement-activating mouse monoclonal AQP4-IgG. Parenchymal infiltration of netting neutrophils containing C5a ensued with microglial activation and motor impairment but no blood-brain barrier leakage. Motor impairment and neuronal dysfunction both reversed when AQP4-IgG infusion stopped. Two-photon microscopy and electron microscopy–based reconstructions revealed physical interaction of infiltrating neutrophils with microglia. Ablation of either peripheral neutrophils or microglia attenuated the motor deficit, highlighting their synergistic pathogenic roles. Of note, mice lacking complement receptor C5aR1 exhibited reduction in neutrophil infiltration, microglial lysosomal activation, neuronal lipid droplet burden, and motor impairment. Pharmacological inhibition of C5aR1 recapitulated this protection. Immunohistochemical analysis of an NMO patient’s spinal cord revealed disease-associated microglia surrounding motor neurons in nondestructive lesions. Our study identifies neutrophil-derived C5a signaling through microglial C5aR1 as a key early driver of reversible motor neuron dysfunction in the precytolytic phase of NMO.

Authors

Fangfang Qi, Vanda A. Lennon, Shunyi Zhao, Yong Guo, Husheng Ding, Caiyun Liu, Whitney M. Bartley, Tingjun Chen, Claudia F. Lucchinetti, Long-Jun Wu

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Figure 7

Microglia are required for pathogenic AQP4-IgG to upregulate CXCL1 in cultured mouse astrocytes.

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Microglia are required for pathogenic AQP4-IgG to upregulate CXCL1 in cu...
(A) Experimental design of the IgG binding to AQP4 on astrocytes in primary glial cultures established from wild-type and Aqp4–/– pup brains. Microglia were depleted by treating Clodrosome (100 μg/mL) before subculture. Two days after adding IgG or cytokines, CXCL1 production was assessed by immunostaining or ELISA. (B) Immunoblot analysis of WT, Aqp4–/– primary glial cells using IgGs specific for AQP4 and actin (loading control). (C) CXCL1 immunoreactivity was assessed in wild-type and AQP4± cells exposed to a control nonpathogenic monoclonal mouse IgG specific for the AQP4 cytoplasmic C-terminal domain (CCD-AQP4-IgG, m5) or a pathogenic extracellular domain–reactive IgG (ECD-AQP4-IgG, m21) or to IFN-γ plus TNF-α cytokines. Astrocytes are identified by AQP4 and GFAP immunoreactivities; microglia by IBA1; DNA is blue (DAPI). (D) Cellular CXCL1 (upper) was quantified from fluorescence intensity in C images; secreted CXCL1 protein levels (lower) were quantified in the glial culture media by ELISA. (E) Experimental design: 2 groups of WT mice were infused continuously via spinal subarachnoid catheter (day 0 to day 7) with pathogenic ECD-AQP4-IgG or ECD-AQP4-IgG mixed with CXCL1-IgG. (F) Motor function of mice in E was assessed by rotarod test; time: F(8, 40) = 1.319, P = 0.2632; treatment: F(1, 5) = 16.38, P = 0.0098; interaction: F(8, 22) = 7.766, P < 0.0001; n = 4–5 mice per group. (G and H) Representative images at day 7 of IgG infusion and quantification of neutrophils (Ly6G+) in lumbar cord parenchyma (n = 4 mice per group). Higher magnifications (left corners) of boxed areas in G show neutrophils (magenta). One-way ANOVA with Tukey’s post hoc in D; n = 4 wells in D; 2-way repeated measures ANOVA with Holm-Šídák post hoc test in F; t test in H.