anti mouse il 6 neutralizing antibody  (R&D Systems)

Journal: Alzheimer's & Dementia

Article Title: Endothelial NAD + depletion drives vascular senescence and neuroinflammation via mtDNA‐cGAS/STING‐CD38 signaling in Alzheimer's disease

doi: 10.1002/alz.71423

Figure Lengend Snippet: NAD + supplementation suppresses cGAS/STING pathway activation in cerebral endothelial cells of APP/PS1 mice. (A) Heatmap of differentially expressed key cGAS/STING pathway‐related genes (such as Cgas , Sting1 , Irf3 ) identified by RNA‐seq of cerebral vessel‐enriched fractions from APPtg and APPtg + NR mice ( n = 3 per group). (B, C) Representative western blot image (B) and densitometric quantification of cGAS, STING, phospho‐TBK1 Ser172 (p‐TBK1), and phospho‐IRF3 Ser396 (p‐IRF3) in cerebral vessel‐enriched fractions from APPwt, APPtg, and APPtg + NR mice (C; n = 6 per group). (D–G) Representative immunofluorescence images of hippocampus and cortex from APPtg and APPtg + NR mice showing CD31 (green) co‐stained with STING (D, red) or cGAS (F, red); quantification of STING (E) and cGAS (G) fluorescence intensity within CD31 + cerebral vessels were shown ( n = 5 or 6 mice per group); nuclei were counterstained with DAPI (blue). (H) qPCR analysis of SASP genes ( Il6 , Tnf , Il1b , Cxcl10 , Cxcl2 ) in cerebral vessel‐enriched fractions from APPtg and APPtg + NR mice ( n = 5 per group). (I) ELISA quantification of IL‐6, TNF‐α, and IL‐1β in the culture supernatants of bEnd.3 endothelial cells treated with vehicle control, NR, Aβ, or Aβ + NR ( n = 6 per group). (J) SA‐β‐galactosidase staining of bEnd.3 endothelial cells transfected with control siRNA (si‐Ctrl), Cgas siRNA (si‐ Cgas ), or Sting1 siRNA (si‐ Sting ) followed by Aβ stimulation or vehicle control; representative images show SA‐β‐gal + cells indicated by white arrows, with enlarged insets provided; the percentage of SA‐β‐gal + cells were quantified ( n = 5 per group). Data are presented as mean ± SEM. Statistical analyses were performed using one‐way ANOVA followed by Tukey's multiple comparisons test (C, E, G, I, J) or unpaired two‐tailed Student's t ‐test (H). p ‐values are indicated in the figure.

Article Snippet: For IL‐6 pathway analysis, BV‐2 microglia were incubated with 10 ng/ml anti‐mouse IL‐6 neutralizing antibody (α‐IL‐6; R&D systems, #MAB406) or anti‐mouse IL‐6Rα blocking antibody (α‐IL‐6R; R&D systems, #AF1830) in CM‐containing medium from bEnd.3 cultures.

Techniques: Activation Assay, RNA Sequencing, Western Blot, Immunofluorescence, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Control, Transfection, Two Tailed Test

Journal: Alzheimer's & Dementia

Article Title: Endothelial NAD + depletion drives vascular senescence and neuroinflammation via mtDNA‐cGAS/STING‐CD38 signaling in Alzheimer's disease

doi: 10.1002/alz.71423

Figure Lengend Snippet: NAD + supplementation suppresses cGAS/STING activation by enhancing mitochondrial function and preventing cytosolic mtDNA leakage. (A) Quantification of mitochondrial membrane potential using JC‐1 staining in bEnd.3 endothelial cells treated with Aβ, Aβ + NR, or control conditions; representative images are shown in Figure ( n = 5 per group). (B, C) Flow cytometric analysis of intracellular ROS levels in bEnd.3 cells under indicated treatments ( n = 4 per group). (D) qPCR quantification of cytosolic mitochondrial DNA (mtDNA; D‐loop , Non‐Numt , Cox1 ) and nuclear DNA (nDNA; Tert , B2m ) in cerebral vessel‐enriched fractions isolated from APPwt, APPwt + NR, APPtg, and APPtg + NR mice ( n ≥5 per group). (E, F) Representative immunofluorescence images (E) and quantification (F) of co‐localization of CD31 (green) and oxidative DNA damage marker 8‐OHdG (red) in hippocampal and cortex of APPtg and APPtg + NR mice; nuclei were counterstained with DAPI (blue) ( n ≥5 mice per group). (G) Quantification of cytosolic mtDNA and nDNA levels in bEnd.3 cells transfected with siRNA targeting control (si‐Ctrl), Cgas (si‐ Cgas ), or Sting1 (si‐ Sting ) followed by Aβ treatment ( n = 4 per group). (H) Quantification of cytosolic mtDNA and nDNA levels in bEnd.3 cells treated with Aβ, Aβ + mtDNA depletion (ddC), or Aβ + ddC + NR ( n = 4 per group). (I) Relative mRNA expression of SASP‐related cytokines (IL‐6, TNF‐α, IL‐1β, CXCL10, CXCL2) under the same treatment conditions as in (H) ( n = 4 per group). (J, K) Western blot analysis (J) and quantification (K) of cGAS/STING pathway components (cGAS, STING, p‐TBK1, p‐IRF3) and tight junction proteins (ZO‐1, Occludin) in bEnd.3 cells under treatments with Aβ, Aβ + ddC, and Aβ + ddC + NR ( n = 4 per group). Data are presented as mean ± SEM. Statistical significance was assessed using one‐way ANOVA followed by Tukey's multiple comparisons test. P ‐values are indicated in the figure.

Article Snippet: For IL‐6 pathway analysis, BV‐2 microglia were incubated with 10 ng/ml anti‐mouse IL‐6 neutralizing antibody (α‐IL‐6; R&D systems, #MAB406) or anti‐mouse IL‐6Rα blocking antibody (α‐IL‐6R; R&D systems, #AF1830) in CM‐containing medium from bEnd.3 cultures.

Techniques: Activation Assay, Membrane, Staining, Control, Isolation, Immunofluorescence, Marker, Transfection, Expressing, Western Blot

Journal: Alzheimer's & Dementia

Article Title: Endothelial NAD + depletion drives vascular senescence and neuroinflammation via mtDNA‐cGAS/STING‐CD38 signaling in Alzheimer's disease

doi: 10.1002/alz.71423

Figure Lengend Snippet: NAD + supplementation disrupts IL‐6‐mediated endothelial‐microglial inflammatory crosstalk in AD. (A) Representative immunofluorescence images showing co‐staining of microglial marker Iba1 (red) and endothelial marker CD31 (green) in the cortex and hippocampus of APP/PS1 mice; white arrows indicate perivascular microglia closely associated with cerebral vessels. (B) Quantification of the proportion of perivascular microglia relative to total microglia ( n ≥ 5 per group). (C) Triple immunofluorescence staining of Iba1 (red), CD31 (green), and IL‐6R (gray) to visualize IL‐6R expression in perivascular microglia; yellow arrows indicate IL‐6R‐positive perivascular microglia. (D) Quantification of IL‐6R fluorescence intensity in vessel‐associated microglia ( n ≥5 per group). (E–F) Western blot analysis (E) and densitometric quantification (F) of IL‐6R, JAK1, and phosphorylation levels of STAT3 and NF‐κB p65 in microglia stimulated with conditioned media from bEnd.3 cells treated with vehicle (Con), NR, Aβ, or Aβ + NR ( n = 6 per group). (G–H) Western blot analysis (G) and quantification (H) of IL‐6R, JAK1, and p‐STAT3/p‐NF‐κB p65 in microglia co‐treated with Aβ‐challenged endothelial conditioned medium and isotype IgG, IL‐6‐neutralizing antibody (α‐IL‐6), or IL‐6R‐neutralizing antibody (α‐IL‐6R) ( n = 4 per group). Data are presented as mean ± SEM. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. P ‐values are indicated in the figure.

Article Snippet: For IL‐6 pathway analysis, BV‐2 microglia were incubated with 10 ng/ml anti‐mouse IL‐6 neutralizing antibody (α‐IL‐6; R&D systems, #MAB406) or anti‐mouse IL‐6Rα blocking antibody (α‐IL‐6R; R&D systems, #AF1830) in CM‐containing medium from bEnd.3 cultures.

Techniques: Immunofluorescence, Staining, Marker, Expressing, Fluorescence, Western Blot, Phospho-proteomics

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: Differential Effects of vIL6 and hIL6 on the frequency and distribution of KSHV infection in B cells. Naïve B cells were isolated from 15 unique tonsils and Mock infected or infected with BAC16‐derived KSHV‐WT or KSHV‐ΔK2. Cultures were treated with IL6 neutralizing antibodies or left untreated and analyzed at 3 dpi for the distribution of KSHV infection within B cell lineages (Table ) using the GFP reporter by flow cytometry. (A) percent of viable CD19 + B lymphocytes that were GFP+ in each condition. Two‐way repeated measures ANOVA p = 0.006, F = 10.6 for Nab treatment, post hoc paired T ‐test p = 0.04 comparing no treatment to IL6 neutralization in KSHV‐WT infection. (B) Percent of GFP+ plasmablasts; paired T ‐test p = 0.05 comparing WT to ∆K2 in untreated cultures (C) percent of GFP+ classical memory cells; Two‐way repeated measures ANOVA p = 0.03, F = 5.7 for Nab treatment, p = 0.03, F = 5.9 for virus strain; paired T ‐test p = 0.04 comparing WT to ∆K2 in untreated cultures. (D) Percent of GFP + IL6 + CD19+ cells; paired T ‐test p = 0.03 comparing WT and ∆K2 infection in the untreated cultures. (E) Correlation between total infection (GFP + CD19 + ) and GFP within plasmablast using pearson's method. (F) Correlation between total infection (GFP + CD19 + ) and GFP within classical memory using pearson's method. (G) lack of correlation between total infection (GFP + CD19 + ) and GFP within IL6 + B cells. (H) Percent of GFP + MZ‐like cells; Two‐way repeated measures ANOVA p = 0.006, F = 10.6 for Nab treatment, post hoc paired T ‐test p = 0.03 comparing no treatment to IL6 neutralization in KSHV‐∆K2 infection. (I) Correlation between total infection (GFP + CD19 + ) and GFP within MZ‐like using pearson's method.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Isolation, Derivative Assay, Flow Cytometry, Neutralization, Virus

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: vIL6 impacts hIL6 expression in B cell subsets. Naïve B cells were isolated from 15 unique tonsils and Mock infected or infected with BAC16‐derived KSHV‐WT or KSHV‐ΔK2. Cultures were treated with IL6 neutralizing antibodies or left untreated. At 3 dpi supernatants were analyzed for secreted hIL6 and cells were analyzed for hIL6 expression within B cell subsets by ICCS. (A) Frequency of IL6 + B cells. Two‐way repeated measures ANOVA p = 0.002, F = 7.6 for virus strain, post hoc paired T ‐test p = 0.006 comparing KSHV‐WT to ∆K2 in the no treatment condition. (B) Secreted hIL6 showed the same statistical effect as IL6+ within B cells ( p = 0.01). (C) Frequency of IL6+ within B cell subsets with the following significant effects. Plasma cell: Two‐way repeated measures ANOVA p = 0.02, F = 4.4 for virus strain, post hoc paired T ‐test p = 0.05 comparing KSHV‐WT to ∆K2 in the hIL6 neutralized condition. Double negative memory: paired T ‐test p = 0.0 comparing KSHV‐WT to ∆K2 in the no treatment condition. Naive: Two‐way repeated measures ANOVA p = 0.03, F = 3.9 for virus strain, post hoc paired T ‐test p = 0.02 comparing KSHV‐WT to ∆K2 in the no treatment condition. Transitional: Two‐way repeated measures ANOVA p = 0.003, F = 7.3 for virus strain, post hoc paired T ‐test p = 0.04 comparing KSHV‐WT to ∆K2 in the no treatment condition. Germinal Center: Two‐way repeated measures ANOVA p = 0.002, F = 7.7 for virus strain, post hoc paired T ‐tests p = 0.02 comparing KSHV‐WT to ∆K2 in the hIL6 neutralized condition, p = 0.01 comparing KSHV‐WT to ∆K2 in the no treatment condition.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Expressing, Isolation, Infection, Derivative Assay, Virus, Clinical Proteomics

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: vIL6 and hIL6 differentially alter secretion of TNF‐α, BAFF and IL‐10 during KSHV infection. Supernatants from 11 unique tonsils infected and treated as in experiments described in Figure were harvested at 3 dpi and clarified of cellular debris by centrifugation. Concentrations of 13 cytokines related to B cell activation and differentiation were determined using the Biolegend Legendplex Human B cell panel for each supernatant. Cytokines significantly altered by experimental parameters were (A) BAFF with 2‐way repeated measures ANOVA showing significant interaction of virus infection and hIL‐6 neutralization ( p = 0.03, F = 4.4) and significantly different pairwise comparison via T‐test comparing Mock+IL6 NAb and KSHV‐∆K2 + IL6 Nab ( p = 0.039). (B) TNF‐α with 2‐way repeated measures ANOVA showing significant effect of virus condition ( p = 0.05, F = 3.6) and significant pairwise comparisons via T ‐test comparing Mock and KSHV‐WT ( p = 0.008) and KSHV‐WT with KSHV‐∆K2 ( p = 0.01) with no hIL6 neutralization; (C) IL‐10 with significant pairwise comparisons via T‐test comparing Mock and KSHV‐WT ( p = 0.02) with no hIL6 neutralization.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Centrifugation, Activation Assay, Virus, Neutralization, Comparison

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: vIL6 and hIL6 cooperatively support B cell viability during KSHV infection but have differential effects on subset frequencies. Naïve B cells were extracted from a distinct set of 15 tonsils and subjected to infection with either Mock, KSHV‐WT, or KSHV‐ΔK2. The cultures were either supplemented with IL6‐neutralizing antibodies or remained untreated. At 3dpi, the cultures were examined by flow cytometry to assess B cell viabilities and the frequencies of B cell subsets. (A) Percent of viable CD19 + . Two‐way repeated measures ANOVA p = 0.01, F = 5.3 for virus strain, p = 0.04, F = 5.5 for hIL6 neutralization and p = 0.03, F = 3.9 for the interaction of the two variables. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.01 comparing no treatment to hIL6 neutralization in Mock cultures, p = 0.01 comparing Mock with KSHV‐WT in untreated cultures and p = 0.004 comparin Mock with ∆K2 in untreated cultures. (B) Frequency of naïve B cells Two‐way repeated measures ANOVA p = 0.05, F = 4.7 for hIL6 neutralization and p = 0.04, F = 3.6 for the interaction of virus and hIL6 neutralization. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.018 comparing Mock to ∆K2 in the no treatment condition and p = 0.01 comparing untreated to hIL6 neutralization in Mock infected cultures. (C) Frequency of germinal center B cells. Two‐way repeated measures ANOVA p = 0.03, F = 4.1 for virus strain. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.019 comparing Mock to ∆K2 in the no treatment condition. (D) Frequency of plasmablasts. Two‐way repeated measures ANOVA p = 0.00006, F = 14.1 for virus strain and p = 0.001, F = 9.0 for the interaction of virus and hIL6 neutralization. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.03 comparing Mock and WT, p = 0.03 comparing Mock and ∆K2 in the no treatment conditions and p = 0.002 comparing Mock and WT, p = 0.04 comparing Mock and ∆K2 and p = 0.004 comparing WT and ∆K2 in the hIL6 neutralized conditions.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Flow Cytometry, Virus, Neutralization

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: hIL6 and vIL6 have separable effects on the differentiation of plasmablast and germinal center B cells from naïve B cells during infection. I B cells loaded with tracking dye before infection and coculture with unlabeled lymphocyte fractions to track differentiation of naïve B cells over time during Mock, KSHV‐WT or KSHV‐∆K2 infection with or without neutralization of hIL6. At 3 dpi cells were harvested and analyzed for B cell subset markers, KSHV infection (GFP+ populations) and differentiation (dye+ populations). (A) proportional distribution of dye+ B cells amongst B cell subsets (B) frequency of undifferentiated naïve B cells. Two‐way repeated measures ANOVA p = 0.02, F = 3.6 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.031 comparing Mock to ∆K2 in the untreated condition. (C) Frequency of GC B cells differentiated from naïve. Two‐way repeated measures ANOVA p = 0.02, F = 3.6 for virus strain, p = 0.03, F = 3.8 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.039 comparing Mock to ∆K2 and p = 0.035 comparing WT to ∆K2 in the untreated conditions. (D) Frequency of plasmablast differentiated from naïve. Two‐way repeated measures ANOVA p = 0.00004, F = 10.2 for virus strain, p = 0.05, F = 3.9 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.012 comparing Mock to ∆K2 and p = 0.014 comparing Mock to WT in the untreated conditions, p = 0.007 comparing Mock to WT and p = 0.011 comparing WT to ∆K2 in the hIL6 neutralized conditions. (E) Correlation between differentiation of GCB and total frequency of GCB using pearson's method. (F) Correlation between differentiation of plasmablast and total frequency of plasmablast via pearson's method. (G) Correlation between differentiation of GCB and IL6 + GCB within each virus condition using pearson's method. (H) Correlation between GCB differentiation and total KSHV infection using pearson's method. (I) Correlation between differentiation of GCB and KSHV infection of plasmablast using pearson's method. (J) Correlation between differentiation of GCB and KSHV infection of classical memory using pearson's method.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Neutralization, Virus

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: KSHV infection drives functional maturation of B cells during ex vivo infection, but vIL6 suppresses differentiation of IgG+ plasma cells during KSHV infection. Naive B cells loaded with tracking dye before infection and coculture with unlabeled lymphocyte fractions to track differentiation of naïve B cells over time during Mock, KSHV‐WT or KSHV‐∆K2 infection with or without neutralization of hIL6. At 3 dpi cells were harvested and analyzed for B cell subset markers including Ig isotype, KSHV infection (GFP+ populations) and differentiation (dye+ populations). (A) Frequency of differentiated IgG+ B cells normalized to each sample's mock value. Two‐way repeated measures ANOVA conducted before data normalization: p = 0.00008, F = 15.4 for virus strain, p = 0.04, F = 5.1 for hIL6 neutralization and p = 0.002, F = 7.4 for interaction of virus strain and hIL6 neutralization. (B) frequency of differentiated IgG+ plasma cells. Paired T ‐test p = 0.03 comparing WT and ∆K2 in the untreated condition. (C) Concentrations of IgG isotypes at 3 dpi were determined in clarified supernatants from eight samples via Legendplex immunoassay. Two‐way repeated measures ANOVA showed significant effect of virus strain on supernatant concentrations of IgG1 ( p = 0.036, F = 6.4) and significant effect of virus strain ( p = 0.04, F = 5.99) and significant interaction of virus strain and IL6 neutralization ( p = 0.04, F = 6) on supernatant concentrations of IgG3.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Functional Assay, Ex Vivo, Clinical Proteomics, Neutralization, Virus