Journal: Frontiers in Immunology

Article Title: Microglial Nox2 Plays a Key Role in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

doi: 10.3389/fimmu.2021.638381

Figure Lengend Snippet: Nox2-deficient group presented similar profile of peripheral immune response as Nox2-competent group in spleen and cervical lymph nodes on disease peak (17 dpi). (A) the T cell profiles of IFNγ (T H 1), IL-17A (T H 17), IL-10, and Foxp3 (Treg)-producing cells from splenocytes (spleen) and cervical lymph nodes (CLN) respectively between W and K groups (n = 5 each group). (B) the myeloid cell profiles (CD11b + CD45 hi ) of neutrophils (Ly6G + MHC II − ), monocytes (Ly6G - MHC II + ), and cell count of pathogenic monocytes (Ly6G - MHCII + Ly6C + CCR2 + ) from spleen and CLN respectively between W and K groups (n = 5 each group). p value: ns, not significant.

Article Snippet: 3-NT antibody (1:1,500; Abcam, catalog number: ab61392), in combination with antibody specific for CD11b (1:1,500; Bio-Rad, catalog number: MCA711G), ASPA (1:200; Millipore, catalog number: ABN1698), Neu-N (1:1,500; Abcam, catalog number: 177487), Iba-1 (1:1,500; WAKO, catalog number: 019-19741), or GFAP (1:1,500; DAKO, catalog number: 00087880), were used as indicated in the figure legends.

Techniques: Cell Counting

Journal: Frontiers in Immunology

Article Title: Microglial Nox2 Plays a Key Role in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

doi: 10.3389/fimmu.2021.638381

Figure Lengend Snippet: Nox2 deficiency greatly reduces the EAE-elicited invasion of pathogenic immune cells into the CNS on disease peak (17 dpi). (A) the T cell profiles of IFNγ (T H 1), IL-17A (T H 17), IL-10, and Foxp3 (Treg)-producing cells from CNS mononuclear cells between W and K groups (n = 5 each group). (B) the myeloid cell profiles (CD11b + CD45 hi ) of neutrophils (Ly6G + MHC II − ), monocytes (Ly6G − MHC II + ), and (C) the absolute cell counts of pathogenic monocytes (Ly6G − MHCII + Ly6C + CCR2 + ) from CNS mononuclear cells between W and K groups (n = 5 each group). p value: * < 0.05; ns, not significant.

Article Snippet: 3-NT antibody (1:1,500; Abcam, catalog number: ab61392), in combination with antibody specific for CD11b (1:1,500; Bio-Rad, catalog number: MCA711G), ASPA (1:200; Millipore, catalog number: ABN1698), Neu-N (1:1,500; Abcam, catalog number: 177487), Iba-1 (1:1,500; WAKO, catalog number: 019-19741), or GFAP (1:1,500; DAKO, catalog number: 00087880), were used as indicated in the figure legends.

Techniques:

Journal: Frontiers in Immunology

Article Title: Microglial Nox2 Plays a Key Role in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

doi: 10.3389/fimmu.2021.638381

Figure Lengend Snippet: Nox2 deficiency largely reduces the production of oxidative stress and the signal of pNF-κB on disease peak (17 dpi) at spinal cord area. (A) Time-dependent of each parameter in lines as a quantitative manner. The mean fluorescence intensity of whole pixels was measured by Image J. (B) mRNA levels (mean ± SEM) of spinal cord (L4-level section, 0.5 μm thick tissues) at 17 dpi after MOG inoculation were determined by RT-PCR. Cell membrane protein: CD11b and Nox2 , (C) The relationship between 3-NT (3-Nitrotyrosine, oxidative stress marker) signal and Iba-1 (microglia/macrophage), CD45 (leukocytes), CD11b (microglia/macrophage), (D) ASPA (aspartoacylase, active oligodendrocytes), (E) GFAP (astrocytes), (F) NeuN (total neurons). (G) The relationship between pNF-κB (inducible inflammatory transcription factor) and NeuN, CD11b. Genotype: W, Nox2 +/+ ; K, Nox2 −/− . Formalin-fixed frozen section: 10 μm. Spinal cord level: lumbar fourth. (C–F) marginal and ventral area of spinal cord (white matter); (G) ventral area of spinal cord (gray matter). p value: *** < 0.001, ** < 0.01, * < 0.05, ns, not significant.

Article Snippet: 3-NT antibody (1:1,500; Abcam, catalog number: ab61392), in combination with antibody specific for CD11b (1:1,500; Bio-Rad, catalog number: MCA711G), ASPA (1:200; Millipore, catalog number: ABN1698), Neu-N (1:1,500; Abcam, catalog number: 177487), Iba-1 (1:1,500; WAKO, catalog number: 019-19741), or GFAP (1:1,500; DAKO, catalog number: 00087880), were used as indicated in the figure legends.

Techniques: Fluorescence, Reverse Transcription Polymerase Chain Reaction, Membrane, Marker

Journal: Frontiers in Immunology

Article Title: Microglial Nox2 Plays a Key Role in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

doi: 10.3389/fimmu.2021.638381

Figure Lengend Snippet: Cytokine/chemokine and transcriptome profiles of Nox2-deficient microglia. CD11b + CD45 int Tmem119 + microglia were isolated from the CNS tissue of Nox2-competent and deficient mice on disease peak (17 dpi). (A) microglia (CD11b + CD45 int ) count analyzed by flow cytometry (n = 5 each group). (B) Concentrations of cytokines and chemokines released from primary cultured microglia. [n = 4 (W), 2 (K-pooled)]. MOG: 10 μg/ml. p value: * < 0.05; ns, not significant. (C) Relative mRNA levels of cytokine/chemokine genes in isolated microglia as determined by RNAseq. Color scales depict relative fold changes. (D) Significantly enriched KEGG pathways in association with immune response, inflammation, infection, and known EAE related pathways. (E) Correlations of the IFNγ-related pathway with Nox2-competent and deficient microglia transcriptome GSEA. p value: * < 0.05; ns, not significant.

Article Snippet: 3-NT antibody (1:1,500; Abcam, catalog number: ab61392), in combination with antibody specific for CD11b (1:1,500; Bio-Rad, catalog number: MCA711G), ASPA (1:200; Millipore, catalog number: ABN1698), Neu-N (1:1,500; Abcam, catalog number: 177487), Iba-1 (1:1,500; WAKO, catalog number: 019-19741), or GFAP (1:1,500; DAKO, catalog number: 00087880), were used as indicated in the figure legends.

Techniques: Isolation, Flow Cytometry, Cell Culture, Infection