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 1

Neutrophils infiltrating and extruding extracellular traps (NETosis) in NMO mouse spinal cord.

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Neutrophils infiltrating and extruding extracellular traps (NETosis) in ...
(A) Experimental design: catheter inserted via cisterna magna into L4 subarachnoid space (yellow circle, Evans blue verification). Subcutaneous osmotic pump continuously infuses AQP4-IgG or control mouse IgG (1.2 μg/d, in 12 μL). Rotarod motor training, days –3, –2, –1; testing, days 0, +1, +2, +3, +4, and +5. Terminal transcardiac perfusion: cord harvested for immunohistochemical and flow cytometric analyses. (B) Cord-infiltrating neutrophils (Cytek analysis), IgG infusion days 1, 3, and 5. Control mice (first panel) infused 3 days with normal mouse IgG. AQP4-IgG recipients: panels 2–4 (3 mice/group). t-SNE, t-distributed stochastic neighbor embedding. (C–E) IgG infusion day 3: representative images (C and D) and quantification (E) of neutrophils (Ly6G+MPO+) in lumbar parenchyma (4 mice/group). Higher magnification box in C shows neutrophils (magenta) and nuclei (blue). (D1 and D2) Higher magnification Ly6G+MPO+ netting neutrophils in AQP4-IgG-infused cord. (F and G) IgG infusion day 3: representative images in lumbar cord; Ly6G+ cluster (top right, control dorsal cord [yellow arrow] is subarachnoid inflammation at catheter site). Higher magnification of F shows neutrophils in AQP4-IgG recipient white matter, in or around a blood vessel surrounded by Cx3cr1GFP+ microglia/macrophages. Ly6G+ neutrophils quantified in cord section; 4 mice/group (G). (H) Representative images: CD31+ blood vessels and Ly6G+ neutrophils in AQP4-IgG recipient mouse lumbar cord, day 3. Higher magnification boxed Ly6G+/CD31+. Higher magnification (split and merged) boxed Ly6G+ and CD31+. 3D Imaris rendering of Ly6G+ neutrophils (green) and CD31+ vessel (red; transparent) in H2. (I) Lumbar cord distribution of NE+ neutrophils in ventral horn, meninges, and dorsal horn, day 5 (4 mice/group). (J) DAPI+ multilobed nucleus (blue) in Ly6G+ neutrophil (red, round- and rod-shape) and cytoplasmic MPO+ granule protein. 3D rendering and segmentation images show morphology of neutrophil’s nucleus. One-way ANOVA, followed by Tukey’s post hoc multiple comparisons test (B). Unpaired t test in E, G, and I.