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Deficiency of Socs3 leads to brain-targeted experimental autoimmune encephalomyelitis via enhanced neutrophil activation and ROS production
Zhaoqi Yan, … , Hongwei Qin, Etty N. Benveniste
Zhaoqi Yan, … , Hongwei Qin, Etty N. Benveniste
Published April 2, 2019
Citation Information: JCI Insight. 2019;4(9):e126520. https://doi.org/10.1172/jci.insight.126520.
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Research Article Inflammation

Deficiency of Socs3 leads to brain-targeted experimental autoimmune encephalomyelitis via enhanced neutrophil activation and ROS production

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Abstract

Dysregulation of the JAK/STAT signaling pathway is associated with multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). Suppressors of cytokine signaling (SOCS) negatively regulate the JAK/STAT pathway. We previously reported a severe, brain-targeted, atypical form of EAE in mice lacking Socs3 in myeloid cells (Socs3ΔLysM), and that this atypical EAE is associated with cerebellar neutrophil infiltration. There is emerging evidence that neutrophils are detrimental in the pathology of MS/EAE; however, their exact function is unclear. Here we demonstrate that neutrophils from the cerebellum of Socs3ΔLysM mice show a hyperactivated phenotype with excessive production of reactive oxygen species (ROS) at the peak of EAE. Neutralization of ROS in vivo delayed the onset and reduced severity of atypical EAE. Mechanistically, Socs3-deficient neutrophils exhibited enhanced signal transducer and activator of transcription 3 (STAT3) activation, a hyperactivated phenotype in response to granulocyte colony–stimulating factor (G-CSF), and upon G-CSF priming, increased ROS production. Neutralization of G-CSF in vivo significantly reduced the incidence and severity of the atypical EAE phenotype. Overall, our work elucidates that hypersensitivity of G-CSF/STAT3 signaling in Socs3ΔLysM mice leads to atypical EAE by enhanced neutrophil activation and increased oxidative stress, which may explain the detrimental role of G-CSF in MS patients.

Authors

Zhaoqi Yan, Wei Yang, Luke Parkitny, Sara A. Gibson, Kevin S. Lee, Forrest Collins, Jessy S. Deshane, Wayne Cheng, Amy S. Weinmann, Hairong Wei, Hongwei Qin, Etty N. Benveniste

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Figure 1

Socs3ΔLysM mice exhibit brain-targeted, atypical EAE with infiltration of hyperactivated neutrophils.

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Socs3ΔLysM mice exhibit brain-targeted, atypical EAE with infiltration ...
EAE was induced in both Socs3fl/fl and Socs3ΔLysM mice, and cerebellar tissue was collected at the peak of disease for further analysis. (A) Demyelination was assessed on day 14 and quantified by Black Gold staining (n = 4). Arrows indicate demyelinated regions. (B–J) Immune cells isolated from the cerebellum on days 13–14 using a Percoll gradient were subjected to surface staining. (B) Overlay of cerebellar-infiltrating neutrophils from Socs3fl/fl and Socs3ΔLysM mice stained for CD11b, CXCR4, CD62L, and CXCR2 (n = 7–8). (C) Relative expression of surface markers of cerebellar-infiltrating neutrophils (n = 7–8). (D) Total number of cerebellar-infiltrating neutrophils (n = 7–8). (E) Degranulation was measured by analyzing the percentage of CD63+ neutrophils on days 13–14 (n = 4). (F–I) Before surface staining, isolated neutrophils were incubated with CM-H2DCFDA (1 μM) at 37°C for 30 minutes. (F) Percentage of ROS-producing neutrophils (n = 7–8). (G and H) ROS production by neutrophils measured as the MFI of CM-H2DCFDA staining (n = 7–8). (I) ROS production by different immune cell types from the cerebellum on days 13–14. Neutrophils (CD45+CD11b+Ly6CloLy6G+), Ly6C+ monocytic cells (Ly6C+ Mo) (CD45+CD11b+Ly6C+Ly6G–), Ly6C– monocytic cells (Ly6C– Mo) (CD45+CD11b+Ly6C–Ly6G–), microglia (CD45loCD11b+), and other leukocytes (CD45+CD11b–). Plot represents 8 individual samples. (J) Superoxide was measured using DHE (n = 4). (K) RNA was isolated from whole cerebellum on day 13, and Hmox1 expression was analyzed by quantitative reverse transcription PCR (qRT-PCR) (n = 4). All error bars represent ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by 2-tailed Student’s t test. MFI, mean fluorescence intensity; DHE, dihydroethidium.

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