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. 2019 Apr 2;5(9):e126520.
doi: 10.1172/jci.insight.126520.

Deficiency of Socs3 leads to brain-_targeted EAE via enhanced neutrophil activation and ROS production

Zhaoqi Yan  1 , Wei Yang  1 , Luke Parkitny  1 , Sara A Gibson  2 , Kevin S Lee  1 , Forrest Collins  1 , Jessy S Deshane  3 , Wayne Cheng  4 , Amy S Weinmann  4 , Hairong Wei  1 , Hongwei Qin  1 , Etty N Benveniste  1
Affiliations

Deficiency of Socs3 leads to brain-_targeted EAE via enhanced neutrophil activation and ROS production

Zhaoqi Yan et al. JCI Insight. .

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), which 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 hyper-activated 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 exhibit enhanced STAT3 activation, a hyper-activated phenotype in response to 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.

Keywords: Autoimmunity; Inflammation; Multiple sclerosis; Neutrophils; Signal transduction.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Socs3ΔLysM mice exhibit brain-_targeted, atypical EAE with infiltration of hyperactivated neutrophils.
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.
Figure 2
Figure 2. ROS plays an important role in the pathology of atypical EAE.
EAE was induced in Socs3ΔLysM mice. Beginning on day 7, ROS scavenger cocktail (FeTPPS 20 mg/kg, PBN 50 mg/kg, and EUK-134 15 mg/kg) was administered i.p. twice per day for 5 days. (A) Atypical EAE score of mice treated with ROS scavenger cocktail (n = 14) or vehicle control (n = 14). Mice that did not develop EAE (classical or atypical) were excluded. (B) Demyelination was assessed on day 14 and quantified by Black Gold staining (n = 3). Arrows indicate demyelinated regions. (C) On days 13 to 14, immune cells from the cerebellum were isolated by Percoll gradient, and the frequencies of microglia (CD45loCD11b+), neutrophils (CD45+CD11b+Ly6CloLy6G+), Ly6C+ monocytic cells (CD45+CD11b+Ly6C+Ly6G–), Ly6C– monocytic cells (CD45+CD11b+Ly6C–Ly6G–), and CD3+ T cells (CD45+CD11b–CD3+) were determined (n = 6). (D) RNA was isolated from whole cerebellum on day 13, and Hmox1 expression was analyzed by qRT-PCR (n = 5). All error bars represent ± SEM. *P < 0.05, and **P < 0.01 by Mann-Whitney rank-sum test (A) or 2-tailed Student’s t test (B–D).
Figure 3
Figure 3. Socs3 deficiency promotes G-CSF hypersensitivity in neutrophils via JAK1 activation.
(A) Bone marrow neutrophils were isolated from C57BL/6 mice and stimulated with G-CSF (10 ng/ml), IL-6 (100 ng/ml), IL-23 (10 ng/ml), or IFN-γ (10 ng/ml) for 2 hours. Expression of Socs3 mRNA was analyzed by qRT-PCR (n = 4). (B–E) Bone marrow neutrophils were isolated from Socs3fl/fl or Socs3ΔLysM mice (n = 3). (B and C) Neutrophils were stimulated with G-CSF (10 ng/ml) or IL-6 (100 ng/ml) for 2 hours followed by intracellular staining for phosphorylated STAT3 (p-STAT3) (Y705). (D and E) Neutrophils were stimulated with G-CSF (10 ng/ml) for 8 hours, and expression of surface markers was analyzed. (F) Bone marrow neutrophils isolated from Socs3ΔLysM mice were pretreated with AZD1480 (25 μM) or PF8041 (25 μM) for 2 hours and then stimulated with G-CSF (10 ng/ml) for 8 hours, followed by surface staining. Plots are representative of 3 independent experiments. All flow cytometry plots (B–F) were gated on live, single CD45+CD11b+Ly6G+ neutrophils. All error bars represent ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by 1-way ANOVA (A) or 2-tailed Student’s t test (C and E). MFI, mean fluorescence intensity.
Figure 4
Figure 4. G-CSF induces a unique gene expression profile in Socs3-deficient neutrophils.
Bone marrow neutrophils isolated from Socs3fl/fl and Socs3ΔLysM mice were stimulated with G-CSF (10 ng/ml) for 8 hours, followed by mRNA extraction and RNA-Seq (n = 3). (A) Heatmap of differentially expressed genes (DEGs) among the 4 groups. (B) Venn diagram depicting DEGs between Socs3fl/fl and Socs3-deficient neutrophils in response to G-CSF. (C) DEGs between Socs3-deficient neutrophils and Socs3fl/fl neutrophils upon G-CSF treatment were ranked based on the adjusted P value and log2 fold change. GSEA illustrating upregulated and downregulated pathways in Socs3-deficient neutrophils in response to G-CSF. (D) Upregulated genes from selected pathways. (E) Bone marrow neutrophils isolated from Socs3fl/fl and Socs3ΔLysM mice were stimulated with G-CSF (10 ng/ml) for 8 hours, followed by mRNA extraction and qRT-PCR assay to determine mRNA expression of the indicated chemokines (n = 3–6). All error bars represent ± SEM. **P < 0.01 by 2-tailed Student’s t test.
Figure 5
Figure 5. Socs3-deficient neutrophils are functionally hyperactivated in vitro upon G-CSF priming.
Bone marrow neutrophils were isolated from Socs3fl/fl or Socs3ΔLysM mice and primed with G-CSF (10 ng/ml) for 12 hours. (A–C) Neutrophils were incubated with (A) PMA (10 ng/ml), (B) PMA plus TNF-α (10 ng/ml), or (C) PMA plus GM-CSF (10 ng/ml), and production of ROS was measured by a luminol assay. Plot represents 4 to 5 independent experiments. (D) G-CSF–primed neutrophils were stimulated with LPS (50 ng/ml) for 1 hour, and degranulation was determined by surface staining of CD63 (n = 3). Flow cytometry plots were gated on live, single CD45+CD11b+Ly6G+ neutrophils. All error bars represent ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by 2-tailed Student’s t test (B–D).
Figure 6
Figure 6. G-CSF neutralization suppresses brain-_targeted, atypical EAE.
EAE was induced in Socs3ΔLysM mice, and from day 7, anti–G-CSF Ab (20 μg/mouse) or isotype control Ab (20 μg/mouse) was administered i.p. every other day until day 13. (A) On day 12, plasma samples were collected and G-CSF levels determined by ELISA (n = 3). (B) Atypical EAE scoring of mice treated with anti–G-CSF Ab (n = 11) or isotype control Ab (n = 14). Mice that did not develop EAE (classical or atypical) were excluded. (C) Demyelination was assessed and quantified by Black Gold staining (n = 5). Arrows indicate demyelinated regions. (D, E, and G) Immune cells were isolated from the cerebellum 14 days after immunization (n = 5–8). (D) Frequencies of microglia (CD45loCD11b+), neutrophils (CD45+CD11b+Ly6CloLy6G+), Ly6C+ monocytic cells (CD45+CD11b+Ly6C+Ly6G–), Ly6C– monocytic cells (CD45+CD11b+Ly6C–Ly6G–), and CD3+ T cells (CD45+CD11b–CD3+) from the cerebella of mice treated with isotype Ab and anti–G-CSF Ab were determined. (E) Total numbers of ROS-producing neutrophils in the cerebella from mice treated with isotype Ab and anti–G-CSF Ab. (F) RNA was isolated from whole cerebellum, and Hmox1 expression was analyzed by qRT-PCR (n = 4). (G) Degranulation of cerebellar-infiltrating neutrophils from mice treated with isotype Ab or anti–G-CSF Ab was determined by surface expression of CD63. All error bars represent ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, and ns, not significant, by Mann-Whitney rank-sum test (B) or 2-tailed Student’s t test (A and C–G).
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