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 8

Disease-associated microglia interact with motor neurons in an NMO mouse model.

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Disease-associated microglia interact with motor neurons in an NMO mouse...
(A) Representative immunostained images of motor neuron–associated (ChAT+) microglia (IBA1+) in ventral gray matter of mice infused for 3 days with IgG infusion. Boxed areas are enlarged on the right. 3D rendering of boxed area in AQP4-IgG mice is shown as split confocal channels (lower right) and merged (lower center). (B and C) Quantification of microglia-neuron co-colocalization area (B) and volume (C) in A (n = 6 mice per group in B; n = 1,526 and 1,272 microglial contacts, respectively, for Ctrl-IgG and AQP4-IgG mice). (D) ImageJ analysis shows the percentage area occupied by Galectin-3 within contacting microglia is greater than in noncontacting microglia in lumbar cord of the 2 experimental groups. (E) Overlaid confocal images show microglial P2Y12 receptor (yellow), Galectin-3 marker of DAMs (cyan) contacting neurons in lumbar ventral gray matter of AQP4-IgG-infused mice, in contrast with control-IgG–infused mice. (F) Percentage of DAMs (IBA1+Gal3+) contacting neurons is significantly increased after AQP4-IgG infusion. (G) The enhanced expression of P2Y12 receptor by IBA1+Gal3+ DAMs (38.5%) contacting neurons implicates P2Y12 in the contact mechanism. (H) Case 1 (left) immunohistochemistry reveals microglia (P2Y12+, brown) associated with neurons in this early stage in spinal cord ventral gray matter lesion of an NMO patient. Arrows identify microglia-neuron contact sites. Case 2 (middle and right) immunohistochemistry combined with immunofluorescence reveals Galectin-3 (brown; IHC) expressed in neuron-associated IBA1+ microglia (green; IF) in an early lesion of a second NMO patient’s spinal cord ventral gray matter. Magenta arrows identify microglia (Galectin-3+IBA1+) associated with motor neurons; blue arrows identify infiltrated neutrophils in this area. Statistics in B–D used t test.