Journal: Frontiers in Immunology

Article Title: Siponimod (BAF312) Activates Nrf2 While Hampering NFκB in Human Astrocytes, and Protects From Astrocyte-Induced Neurodegeneration

doi: 10.3389/fimmu.2020.00635

Figure Lengend Snippet: Characterization of iPSC-derived astrocytes. (A) Phase contrast images showing human iNPC (left panel) and human iAstrocytes (right panel). (B,C) Representative immunofluorescence stainings for GFAP, S100β, nestin, vimentin (B) and S1P1, S1P3, IL1R and IL17R (C) in human iAstrocytes. DAPI was used for nuclear staining. (D,E) Percentage of cells positive for astrocyte markers at two timepoints during differentiation. Reported quantifications were performed on three different human iAstrocytes preparations from the same iNPC cell line. Bars represent SEM. Same observations were recorded in human iAstrocytes from a second iNPC cell line. Scale bar = 30 μm.

Article Snippet: The following primary antibodies were used: rabbit anti-GFAP (Agilent), mouse anti-nestin (Merck Millipore), mouse anti-vimentin (Abcam), rabbit anti-S100β (Abcam), rabbit anti-EDG1 (Santa Cruz Biotechnology), rabbit anti-EDG3 (Santa Cruz Biotechnology), mouse anti-IL1R (R&D), rabbit anti-IL17R (Santa Cruz biotechnology), rabbit anti- NFκB p65 (Abcam), rabbit anti-GLAST (Abcam), guinea pig anti-GLT1 (Merck Millipore), rabbit anti-Nrf2 (Abcam), mouse anti Neuronal Class III β-Tubulin (Covance).

Techniques: Derivative Assay, Immunofluorescence, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Systemic Antibiotic Therapy Reduces Circulating Inflammatory Dendritic Cells and Treg-Th17 Plasticity in Periodontitis.

doi: 10.4049/jimmunol.1900046

Figure Lengend Snippet: Increased expression of IL-1R1 and IL-6Rα in CD4+Foxp3+ T cells in PD patients. CD4+ T cells were stimulated as in figure 3 in the presence and absence of IL-1β+IL-6. (A and B) Frequencies of C4+Foxp3+ T cells expressing IL-1R1 was increased in non-AB (n = 6–9) patients compared to healthy controls (HC) (n = 8) and AB (n = 5–6) subjects at day6 (A) and final (B) time points (Data are mean ± SEM, *p<0.05, **p<0.01, ***p<0.001). (C and D) Graphs depicts the Spearman’s positive correlation of Foxp3+IL-1R1+ population with %IL17A+Foxp3+ T cells (C) and Th17 cells (D) at final time point in response to IL1-β and IL-6 in non-AB group (n=6). (E and F) Graph shows the percentage of C4+Foxp3+ T cells expressing IL6Rα in healthy controls (HC) (n = 8), non-AB (n = 6–9), and AB (n = 5–6) subjects at day6 (E) and final (F) time points (Data are mean ± SEM, *p<0.05, **p<0.01). (G) Graph depicts the Spearman’s positive correlation between the Foxp3+IL-6Rα+ population and %IL17A+Foxp3+ T cells at final time point in response to IL1-β and IL-6 in non-AB group (n=9). (H) Increased expression of IL-1R1 in IL-17A+Foxp3+ Treg compared to conventional Treg and Th17 populations in periodontitis (PD) patients (Data are mean ± SEM, *p<0.05, **p<0.01, ***p<0.001). (I) Increased expression of IL-6Rα in IL-17A+Foxp3+ Treg compared to conventional IL17A-Foxp3+ Treg (Data are mean ± SEM, *p<0.05).

Article Snippet: Conjugated antibodies against the following were from R&D systems: RORγT (clone AFKJS-9) and IL-1R1 (Catalog number FAB269F).

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-1β induces the rapid secretion of the antimicrobial protein IL-26 from Th17 cells

doi: 10.4049/jimmunol.1900318

Figure Lengend Snippet: (A) Sorting scheme used to isolate helper T cell subsets, a representative donor is shown, n = 3. (B) Isolated Th1, Th2, and Th17 cells were stimulated with IL-1β for 48 hours and cell free supernatants were analyzed for IL-26 secretion by ELISA, n = 3. (C) A representative sorting scheme for isolating IL-1RI+ and IL-1RI- Th17 cells, n= 3. (D, E) IL-1RI+ and IL-1RI- Th17 cells were stimulated with 100 ng/ml IL-1β or 5 µl/ml anti-CD3/CD28 for 9 hours before IL-26 (D) and IL-17A (E) was measured in cell free supernatants by ELISA, n = 3. (B,D) Data are represented as mean ± SEM, statistics calculated using one-way ANOVA with Tukey’s post-test (B, D, E). **p ≤ 0.01. *** p ≤ 0.001

Article Snippet: In some experiments, memory CD4 + T cells were incubated overnight with 1 nM recombinant human IL-2 and stained additionally with antibodies against APC-IL-1RI (R&D Systems, polyclonal goat anti-human) to sort IL-1RI + Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI + ) and IL-1RI - Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI - ).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-1β induces the rapid secretion of the antimicrobial protein IL-26 from Th17 cells

doi: 10.4049/jimmunol.1900318

Figure Lengend Snippet: (A) Unsupervised principal component analysis of IL-1RI+ and IL-1RI- Th17 cells from 3 donors treated with IL-1β, anti-CD3/CD28, or media as a control. Numbers within symbols indicate the donor. (B) Hierarchical clustering of the samples. (C) Differential gene expression in untreated IL-1RI+ Th17 cells vs. IL-1RI- Th17 cells is represented in a volcano plot and some of the most differentially expressed genes are named and highlighted with black dots. (D) Genes with a (FC) ≥ 1.5 and p-adj ≤0.05 after treatment of IL-1RI+ Th17 cells with IL-1β vs media are plotted based on their normalized counts after IL-1β or anti-CD3/CD28 treatment. The line y = x was plotted on the graph to show genes with greater relative counts in each condition. Genes in the IPA “Th17 cell activation” canonical pathway and CXCL8 are highlighted with black dots. (E) Memory CD4+ T cells were treated with 100 ng/ml IL-1β and 5 µl/ml anti-CD3/CD28 for 48 hours before supernatants were collected and IL-8 protein concentrations were measured by ELISA, n = 4. Data are represented as mean ± SEM, statistics calculated using one-way ANOVA with Tukey’s post-test. *** p ≤ 0.001.

Article Snippet: In some experiments, memory CD4 + T cells were incubated overnight with 1 nM recombinant human IL-2 and stained additionally with antibodies against APC-IL-1RI (R&D Systems, polyclonal goat anti-human) to sort IL-1RI + Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI + ) and IL-1RI - Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI - ).

Techniques: Control, Gene Expression, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-1β induces the rapid secretion of the antimicrobial protein IL-26 from Th17 cells

doi: 10.4049/jimmunol.1900318

Figure Lengend Snippet: “Th17 cell activation” pathway genes significantly upregulated by IL-1β treatment of IL-1RI + Th17 cells. The IPA canonical pathway “Th17 cell activation” was the most significantly enriched canonical pathway within our dataset (p = 2.51 × 10 −17 ). This pathway contained 90 genes, 19 of which, or 21%, were contained in our dataset and are displayed in the table.

Article Snippet: In some experiments, memory CD4 + T cells were incubated overnight with 1 nM recombinant human IL-2 and stained additionally with antibodies against APC-IL-1RI (R&D Systems, polyclonal goat anti-human) to sort IL-1RI + Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI + ) and IL-1RI - Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI - ).

Techniques: Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-1β induces the rapid secretion of the antimicrobial protein IL-26 from Th17 cells

doi: 10.4049/jimmunol.1900318

Figure Lengend Snippet: (A, B) Genes with a log2(FC)≥ 0.58 and p-adj ≤0.05 after treatment of IL-1RI+ Th17 cells with IL-1β (A) or anti-CD3/CD28 (B) were analyzed for enrichment of upstream transcription factors using Enrichr and plotted with their -log10(p-adj). C. Memory CD4+ T cells were stimulated with either 20 ng/ml IL-1β or 5 µl/ml anti-CD3/CD28 for 24 hours, with or without 1 hour pretreatment with 10µM Bay 11–7082, an NF-κB activation inhibitor. IL-26 secretion into cell supernatants was measured by ELISA, n = 3. Data are represented as mean ± SEM, statistics calculated using one-way ANOVA with Tukey’s post-test. *p ≤ 0.05, *** p ≤ 0.001.

Article Snippet: In some experiments, memory CD4 + T cells were incubated overnight with 1 nM recombinant human IL-2 and stained additionally with antibodies against APC-IL-1RI (R&D Systems, polyclonal goat anti-human) to sort IL-1RI + Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI + ) and IL-1RI - Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI - ).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay