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 3

Microglial activation and motor impairment by AQP4-IgG requires CNS-infiltrating neutrophils.

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Microglial activation and motor impairment by AQP4-IgG requires CNS-infi...
(A) Timeline for injecting neutrophil-depleting anti-Ly6G-IgG or isotype control-IgG (100 mg/kg, i.p.), inserting lumbar subarachnoid catheter, infusing AQP4-IgG, and rotarod testing. (B) Flow cytometry confirms neutrophil ablation efficiency (percentage CD45+CD11b+Gr1+MPO+ cells among peripheral CD45+CD11b+ cells). (C) (Upper) t-SNE analysis of CD45+ immune cell subtypes from lumbar spinal cords of control and neutrophil-depleted mice. (Lower) Quantification of data in B (3 mice/group). (D) Representative confocal images of neutrophils in lungs of mice receiving neutrophil-depleting anti-Ly6G-IgG or isotype control-IgG (3 mice/group). (E) Microglial activation, reflected by Cx3cr1GFP signal, in corresponding lumbar cord regions of mice without and with neutrophil ablation (by Ly6G-IgG or isotype control-IgG) after 3 days’ infusion with normal control mouse IgG or AQP4-IgG. (F) Quantification of microglia-occupied areas in E (n = 4–5 mice per group). (G) Motor function, reflected by rotarod test, in neutropenic mice (anti-Ly6G-IgG–treated) and non-neutrophil-ablated (isotype control-IgG–treated) during 5 days’ infusion of AQP4-IgG or normal control mouse IgG (0.1 μg/μL; time: F(2.415, 28.98) = 4.838, P = 0.0113; treatment: F(2, 12) = 12.46, P = 0.0012; interaction: F(10, 60) = 7.100, P < 0.0001; n = 5 mice per group). (H) Experimental design: WT mice were continuously infused with Ctrl-IgG or AQP4-IgG by osmotic pumps for 7 days from day 0; infusion was discontinued at day 8. (I) Motor impairment worsened progressively in AQP4-IgG recipients, with nadir at day 8. Continued rotarod testing for another 3 weeks showed progressive motor recovery from day 8. (J) Correlations between microglial activation state (lumbar microglial area) and latency to fall in rotarod test. Simple linear regression (1 dot represents 1 mouse at day 3 of IgG infusion). Statistics: C used t test; Tukey’s post hoc multiple comparisons test (1-way ANOVA) in F; 2-way repeated measures ANOVA with Holm-Šídák post hoc test in G and I.