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
View: Text | PDF
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

×

Figure 6

C5ar1 deficiency ameliorates neuronal dysfunction and lipid droplet accumulation in motor neurons.

Options: View larger image (or click on image) Download as PowerPoint
C5ar1 deficiency ameliorates neuronal dysfunction and lipid droplet acc...
(A) Experimental design: mice were infused continuously with AQP4-IgG using osmotic pumps from day 0 through day 7. (B–D) Quantification of Nissl bodies (B), Nissl area (C), and percentage area occupied by ChAT+ motor neurons in gray matter of lumbar cord of Ctrl-IgG–infused mice at day 3, AQP4-IgG–infused mice at days 3, 14, and 28. (E–H) Representative images of Nissl bodies (red) in ventral gray matter (vGM) neurons of wild-type and C5ar1–/– mice at day 3. Boxed areas are enlarged on the right. (I and J) Numbers and sizes of Nissl body+ neurons in vGM (n = 8 mice per group). (K) Representative motor neuron confocal images and quantification in vGM of WT mice infused with Ctrl-IgG or AQP4-IgG and C5ar1–/– mice infused with AQP4-IgG or control-IgG (not shown). HuD+ (green); ChAT+ (magenta). (L and M) Numbers of ChAT+ motor neurons (MNs) and their percentage among total neurons (HuD+ in vGM) (n = 4 mice per group). (N) Representative images of BODIPY+ lipid droplets (green) in ChAT+ motor neurons (gray) and NeuN+ neurons (gray) in vGM of WT and C5ar1–/– mice infused with AQP4-IgG. 3D reconstruction of BODIPY+ lipid and NeuN+ neurons (right). (O) Quantification of lipid droplet numbers in the cytoplasm of NeuN+ neurons. (P) Representative images of 4-HNE (peroxidative stress marker) in ChAT+ motor neurons. (Q and R) Q, Quantification of 4-HNE and ChAT immunoreactivity intensities across 30 μm neuronal diameter; R, presented as mean ± SEM (n = 20 neurons from 4 mice per group). One-way ANOVA with Tukey’s post hoc in B–D, L, M, and O; 2-way (treatment × genotyping) ANOVA with Holm-Šídák post hoc multiple comparisons test in I and J; t test in R.