Journal: Nature medicine

Article Title: Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and CNS inflammation via the aryl hydrocarbon receptor

doi: 10.1038/nm.4106

Figure Lengend Snippet: EAE in GFAP-AhR-deficient or control mice. ( a ) Top: Clinical scores (mean ± s.e.m.; representative out of five independent experiments with n = 10 mice per group; Two-way ANOVA). Bottom: Ratio of RNA abundances in pro-inflammatory cluster from sorted GFAP-AhR-deficient and control astrocytes at the peak of disease (fold change in relative expression as determined by log 2 (GFAP-AhR/Control). Representative of two independent experiments of pooled astrocytes of n = 3 mice per group. ( b ) Absolute number of CNS infiltrating CD11b + Ly-6C hi monocytes as assessed by FACS analysis. n = 5 per group, representative of five independent experiments, Student’s t -test. ( c ) Left two figures: Nanostring analysis of pro-inflammatory gene clusters from sorted CD11b + CD45 lo microglia (left) and CD11b + Ly-6C hi monocytes (right); numbers of GFAP-AhR divided by Control; representative out of two independent experiments of pooled microglia and macrophages of n = 3 mice per group; Student’s t -test. Right three figures: RNA expression of indicated genes in astrocytes sorted from WT and GFAP-AhR mice at peak of disease. ( n = 3, Student’s t -test; normalized to Control Ccl2 ) ( d ) Left: Supernatants of LPS or vehicle stimulated WT or GFAP-AhR-deficient astrocytes were investigated in migration assays using CD11b + Ly6C hi WT monocytes as migrating cells (absolute cell numbers; n = 3; representative of three independent experiments; one-way ANOVA, Tukey’s multiple comparisons test). Right: Migration assay using blocking antibodies as indicated or IL-27R KO macrophages (fold cell numbers; n = 3; representative of three independent experiments; one-way ANOVA within treatment groups, Turkey’s multiple comparisons test). ( e ) Left panel: Sorted CD11b + Ly6C hi monocytes were co-cultured with activated control or GFAP-AhR-deficient astrocytes, re-isolated and gene-expression analyzed by qPCR ( n = 3, representative of two independent experiments; Student’s t -test; normalized to Control Ccl2 in c ). Right graph: Neurotoxicity assay with supernatants from control or GFAP-AhR-deficient astrocytes after activation with LPS or vehicle n = 3, representative of two independent experiments, one-way ANOVA, Tukey’s multiple comparisons test). ( f ) ChIP analysis of binding of NF-kB (p65) to the promoters of Ccl2, Csf2 and Nos2 in Control or GFAP-AhR-deficient astrocytes after activation with LPS. ( n = 3, representative of two independent experiments, one-way ANOVA, Tukey’s multiple comparisons test). ( g ) Schematic of predicted AhR binding sites (XREs) in the SOCS2 promoter (upper graph) and ChIP analysis of AhR binding to the promoter of SOCS2 in astrocytes after stimulation with indicated conditions (lower bar graphs). ( n = 3, representative of two independent experiments, one-way ANOVA, Tukey’s multiple comparisons test; f , g normalized to pCcl2 Control rIgG). ( h ) Relative expression of Socs2 in WT or GFAP-AhR astrocytes after stimulation with LPS (representative out of two independent experiments; one-way ANOVA, Tukey’s multiple comparisons test; normalized to Control Vehicle). ( i ) Western blot detecting NF-kB (p65; left) and quantification (right) of the ratio of nuclear to cytoplasmatic fraction of WT, GFAP-AhR, and SOCS2 −/− astrocytes stimulated with indicated conditions (representative out of three independent experiments, one-way ANOVA, Tukey’s multiple comparisons test). ( j ) qPCR of expression levels of Ccl2 , Csf2 , and Nos2 in Control and SOCS2 −/− astrocytes after stimulation with LPS (representative out of two independent experiments; Student’s t -test; normalized to Control Ccl2 ). Significance levels: * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Human: AHR Hs00169233_m1, CCL2 Hs00234140_m1, CYP1B1 Hs02382916_s1, IFNAR1 Hs01066118_m1, IL6 Hs00985639_m1, IRF9 Hs00196051_m1, MX1 Hs00895608_m1, NOS2 Hs01075529_m1, STAT1 Hs01013996_m1, STAT2 Hs01013123_m1, TNFA Hs01113624_g1.

Techniques: Control, Expressing, RNA Expression, Migration, Blocking Assay, Cell Culture, Isolation, Gene Expression, Activation Assay, Binding Assay, Western Blot

Journal: Nature medicine

Article Title: Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and CNS inflammation via the aryl hydrocarbon receptor

doi: 10.1038/nm.4106

Figure Lengend Snippet: ( a ) qPCR from sorted astrocytes and splenic DCs, macrophages or T cells from naive WT mice treated intranasally with 5.000 IU hIFN-β or PBS daily for 2 days ( n = 3, Student’s t -test; normalized to Astrocytes Ahr ). ( b ) EAE in control or GFAP-AhR mice under intranasal IFN-β treatment. Clinical scores of control (left) or GFAP-AhR-deficient (right) mice (mean ± s.e.m. in left graph; representative out of three independent experiments with n = 10 mice per group; Two-way ANOVA). ( c ) Left panel: RNA abundances from Control and GFAP-AhR astrocytes of indicated genes ( n = 3, representative of two independent experiments, one-way ANOVA, Tukey’s multiple comparison test; normalized to Control Veh Vim ). Middle graph: Quantification of CNS infiltrating CD11b + Ly6C hi inflammatory monocytes; representative out of three independent experiments with n = 10 mice per group; one-way ANOVA, Tukey’s multiple comparison test. Right graph: RNA analysis of pro-inflammatory gene cluster from sorted monocytes; ratio of count numbers of specific treatment group to Control veh; representative out of two independent experiments of pooled monocytes of n = 3 mice per group; one-way ANOVA, Tukey’s multiple comparison test. ( d ) GFAP-AhR-deficient and control animals under indicated treatment starting from day 22 after EAE induction (TDD: Tryptophan depleted diet; Trp: Tryptophan; Clinical scores, mean ± s.e.m.; representative out of two independent experiments with n = 10 mice per group; Two-way ANOVA; Tukey’s multiple comparison test). ( e ) qPCR of Ccl2 and Nos2 in indicated treatment conditions as in ( c ; normalized to Control TDD+Trp Ccl2, one-way ANOVA, Tukey’s multiple comparison test) ( f, g, i ) Clinical scores of WT mice treated with indicated conditions starting from day 22 after EAE induction (mean ± s.e.m.; representative out of two independent experiments with n = 5 mice per group; Two-way ANOVA; Tukey’s multiple comparisons test) ( h ) Left: qPCR of relative mRNA abundances for Ccl2 and Nos2 from astrocytes sorted at day 36 from experimental groups as in f , g , and i ; ( n = 3, representative of two independent experiments; one-way ANOVA followed by Tukey’s multiple comparisons test normalized to Vehicle Ccl2 ). Right: Measurement of I3S in urine samples collected at day 36 of experimental groups as in f, g, i ( n = 3; representative of two independent experiments; one-way ANOVA followed by Tukey’s multiple comparison test). ( j ) SYBR Green qPCR of Lactobacillus reuteri bacterial DNA isolated from fecal samples of indicated groups at day 36 after EAE induction ( n = 4 per group, representative of two independent experiments; one-way ANOVA followed by Tukey’s multiple comparisons test normalized to TDD+Trp). Significance levels: * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Human: AHR Hs00169233_m1, CCL2 Hs00234140_m1, CYP1B1 Hs02382916_s1, IFNAR1 Hs01066118_m1, IL6 Hs00985639_m1, IRF9 Hs00196051_m1, MX1 Hs00895608_m1, NOS2 Hs01075529_m1, STAT1 Hs01013996_m1, STAT2 Hs01013123_m1, TNFA Hs01113624_g1.

Techniques: Control, Comparison, SYBR Green Assay, Isolation

Journal: Nature medicine

Article Title: Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and CNS inflammation via the aryl hydrocarbon receptor

doi: 10.1038/nm.4106

Figure Lengend Snippet: ( a,b ) qPCR analysis of indicated mRNA expression in samples from lesions and normal appearing white matter (NAWM) from individuals with MS, or healthy controls relative to GAPDH ( n = 4 Controls, n = 5 MS NAWM, n = 10 MS Lesion; one-way ANOVA, Tukey’s multiple comparison test; normalized to Control STAT2 ). ( c ) qPCR of RNA levels from human fetal astrocytes treated with IFN-β or vehicle in vitro ( n = 3, representative of two independent experiments, Student’s t -test; normalized to Vehicle AHR ). ( d ) qPCR of pro-inflammatory genes from human fetal astrocytes activated with Poly(I:C) and treated with 3-Indoxylsulfate (I3S) or vehicle. ( n = 3, representative of two independent experiments; Student’s t -test; normalized to Poly(I:C)+I3S NOS2 ). ( e ) Immunofluorescence staining of human white matter brain tissue of active MS lesions for AhR (red), CCL2 (green, left), iNOS (green, right) and GFAP (blue) (Data shown are representative of n = 12 fields from three distinct MS brains) and co-expression of AhR and CCL2, AhR and iNOS, and AhR and GFAP. Scatter graphs (right panel) show the distribution of pixels and extent of colocalization in percentage. Scale bar: 20 μm. ( f ) qPCR analysis of CYP1B1 expression as in a,b . ( g ) Luciferase assay for the determination of absolute amount of AhR ligands in human serum (representative of two independent experiments with 11 Healthy controls, 49 MS; student’s t -test) ( h ) Schematic of tryptophan metabolism (left), and heatmap of median abundances of tryptophan metabolites in serum of healthy controls (HC) and multiple sclerosis patients (right, n = 11 HC, n = 49 MS; Hotelling’s T 2 -test). Significance levels: * P <0.05, ** P <0.01, *** P <0.001, **** P<0.0001, n.s. not statistically significant.

Article Snippet: Human: AHR Hs00169233_m1, CCL2 Hs00234140_m1, CYP1B1 Hs02382916_s1, IFNAR1 Hs01066118_m1, IL6 Hs00985639_m1, IRF9 Hs00196051_m1, MX1 Hs00895608_m1, NOS2 Hs01075529_m1, STAT1 Hs01013996_m1, STAT2 Hs01013123_m1, TNFA Hs01113624_g1.

Techniques: Expressing, Comparison, Control, In Vitro, Immunofluorescence, Staining, Luciferase

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Yolkin Isolated from Hen Egg Yolk as a Natural Immunoregulator, Activating Innate Immune Response in BMDM Macrophages

doi: 10.1155/2020/5731021

Figure Lengend Snippet: Upregulation of iNOS expression in BMDM cells after yolkin treatment. BMDM cells were treated with yolkin (10, 100, and 150 μ g/ml) or LPS (1 μ g/ml) or left untreated for 24 h. The level of iNOS protein was detected in cell lysates by immunoblotting using monoclonal anti-iNOS antibodies (a). Fold change in iNOS levels compared to β -actin (b). Results represent 3-4 independent experiments, and data are presented as mean ± SD. ∗ p ≤ 0.05 vs. control.

Article Snippet: Anti-iNOS monoclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Yolkin Isolated from Hen Egg Yolk as a Natural Immunoregulator, Activating Innate Immune Response in BMDM Macrophages

doi: 10.1155/2020/5731021

Figure Lengend Snippet: Effect of MAPK inhibitors on yolkin-mediated iNOS expression (a) and activation (b). BMDM cells were preincubated with MAPK inhibitors: U0126 for ERK and SP600125 for JNK for 2 h, followed by treatment with yolkin. Cells were lysed and expression of iNOS was determined by Western blot using monoclonal anti-iNOS antibodies (a). Supernatants were used to determine NO concentration by the Griess reaction (b). Results represent three independent experiments and present mean ± SD ( n = 3). ∗ p ≤ 0.05 compared with the control, and ∗∗ p ≤ 0.05 and ∗∗∗ p ≤ 0.05 compared with yolkin.

Article Snippet: Anti-iNOS monoclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Activation Assay, Western Blot, Concentration Assay, Control

Journal: Food & function

Article Title: Suppression of colonic oxidative stress caused by chronic ethanol administration and attenuation of ethanol-induced colitis and gut leakiness by oral administration of sesaminol in mice.

doi: 10.1039/d1fo04120g

Figure Lengend Snippet: Fig. 7 Effects of oral sesaminol administration on the levels of ethanol- induced oxidative-stress proteins (iNOS, CYP2E1, HO-1) and transcrip- tion factor protein (Nrf2) in colonic tissues. (A) WB analyses of iNOS, CYP2E1, HO-1, and β-actin (from top to bottom) in colonic tissue extracts of the mice of groups C, S, E, and ES. Lanes 1–3, group C; lanes 4–6, group S; lanes 7–9, group E; and lanes 10–12, group ES. (B) Protein expression levels of iNOS, CYP2E1, and HO-1 were analysed by WB. The band intensities were measured by densitometry and normalised to β-actin. The relative band intensities (-fold) are shown with the intensity obtained with a subject of group C taken to be 1.0. The data are expressed as means ± SD (n = 5). (C) Nrf2 activation levels were deter- mined by ELISA in colonic tissue extracts, whose protein concentrations were identical (1 μg μL−1) among the four groups. The data are expressed as means ± SD (n = 5). *p < 0.05, **p < 0.01 versus group C, †p < 0.05, ††p < 0.01 versus group S, #p < 0.05 versus group E as assessed by ANOVA with the Tukey–Kramer test.

Article Snippet: The membranes were then incubated with a recommended dilution of one of the following primary rabbit polyclonal antibodies: anti-mouse CYP2E1, anti-mouse inducible nitric oxide synthase (iNOS), anti-mouse zonula occludens-1 protein (ZO-1), anti-mouse occludin, antimouse claudin-1 (each from Abcam), anti-mouse heme oxygenase-1 (HO-1) (Proteintech Group), and anti-mouse β-actin (Genetex; Irvine, CA, USA).

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules

Article Title: Microglia Activated by Excess Cortisol Induce HMGB1 Acetylation and Neuroinflammation in the Hippocampal DG Region of Mice Following Cold Exposure

doi: 10.3390/biom9090426

Figure Lengend Snippet: Acetylation and expression of nuclear factor (NF)-κB signaling pathway components in BV2 cells after cortisol (CORT) treatment (200 μM) for 3 h, as determined by western blotting. ( A ) iNOS, acetyl-p65 (Lys310), p65, SIRT1, and β-actin expression. ( B ) Quantification of expression levels based on inducible nitric oxide synthase (iNOS)/β-actin, acetyl-p65/p65, and silent mating type information regulation 2 homolog (SIRT)1/β-actin ratios. Differences between control and CORT groups were evaluated by one-way analysis of variance. Results are expressed as the mean ± SD ( n = 3) of three independent experiments. *** p < 0.001.

Article Snippet: Approximately 30 μg of total protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (0.22 and 0.45 μm; Millipore, Darmstadt, Germany) that were blocked for 1 h at room temperature in 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST), which were then incubated overnight at 4 °C with antibodies against the following proteins: HMGB1 (#21865-1-AP, 1:3000), GFAP (#16825-1-AP, 1:2000), ionized calcium-binding adapter molecule (IBA)-1 (#10904-1-AP, 1:500), p65 (#10745-1-AP, 1:1000), silent mating type information regulation 2 homolog (SIRT)1 (#66292-1-Ig, 1:3000), brain-derived neurotrophic factor (BDNF); #16806-1-AP, 1:1000), IL-1β (#18985-1-AP, 1:1000), inducible nitric oxide synthase (iNOS) (#12987-1-AP, 1:1000), lamin B1 (#17168-1-AP, 1:3000), histone H3 (#60008-1-lg, 1:15,000), and β-actin (#14395-1-AP, 1:15,000) (all from Proteintech); and extracellular signal-regulated kinase (ERK) (#4695S, 1:1000), phospho-ERK (Thr202/Tyr204) (#9101, 1:1000), c-Jun N-terminal kinase (JNK) (#9252, 1:1000), phospho-JNK (Thr183/Tyr185) (#4668, 1:1000), p38 (#8690, 1:1000), phospho-p38 (Thr180/Tyr182) (#4511, 1:1000), acetyl-p65 (Lys310) (#12629S, 1:1000), and acetyl-histone H3 (Lys9) (#9649, 1:1000) (all from Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Western Blot, Control

Journal:

Article Title: Regulation of macrophage procoagulant responses by the tissue factor cytoplasmic domain in endotoxemia

doi: 10.1182/blood-2006-10-051334

Figure Lengend Snippet: Characterization of bone marrow–derived macrophages (BMMs). (A) BMMs obtained by culture of bone marrow cells from 7- to 10-week-old wild-type, TFΔCT, PAR2−/−, or TFΔCT/PAR2−/− mice were analyzed by flow cytometry for the macrophage surface markers F4/80 and CD11b. (B) Expression of costimulatory molecules on BMMs after overnight stimulation with LPS (1 μg/mL). Isotype-matched control antibody staining (dotted histogram) or specific staining of non–LPS-stimulated (shaded area) or LPS-stimulated (solid line) BMMs are shown; an example of at least 3 independent experiments is given. (C) Morphology of wild-type and TFΔCT BMMs with and without overnight LPS stimulation by phase-contrast microscopy (upper panel) or after phalloidin staining (red) by confocal microscopy; nucleus is stained with ToPro3 (blue) (lower panel).[AU34} (D-F) Effector functions of wild-type or TFΔCT BMMs. Overnight LPS (1 μg/mL) stimulation leads to similar up-regulation of NOS2 by Western blotting (D) and nitric oxide (NO) production (E) determined using the Griess reagent37 (n = 3). (F) FcγR-mediated phagocytosis of IgG-opsonized sheep red blood cells by wild-type and TFΔCT BMMs.

Article Snippet: For detection of nitric oxide synthase 2 (NOS2) activation, BMMs were stimulated with LPS (1 μg/mL) for 18 hours and cell lysates were subjected to Western blotting using anti-NOS2 antibody (SantaCruz Biotechnology, Santa Cruz, CA).

Techniques: Derivative Assay, Flow Cytometry, Expressing, Control, Staining, Microscopy, Confocal Microscopy, Western Blot