Journal: PLoS Biology

Article Title: IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

doi: 10.1371/journal.pbio.1001395

Figure Lengend Snippet: IL-26 was quantified by ELISA in the serums of 26 healthy subjects, 22 RA patients, and 13 patients with other inflammatory arthritis (spondylarthritis, psoriatic arthritis, rhizomelic polyarthritis, and undifferentiated inflammatory arthritis) and in the SFs of 15 RA patients and seven patients with other inflammatory arthritis. Lines correspond to the mean values; * p <0.05 (Mann Whitney test).

Article Snippet: After saturation with PBS containing 2% non-fat dry milk, plates were successively incubated with samples or recombinant human IL-26, with a biotinylated polyclonal goat anti-IL-26 Ab and then with streptavidin-HRP (BD Pharmingen).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: PLoS Biology

Article Title: IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

doi: 10.1371/journal.pbio.1001395

Figure Lengend Snippet: (A) IL-26 expression was analyzed in the synovium of RA patients or trauma patients by immunohistochemistry using an anti-IL-26 mAb (original magnification: ×20). An IgG2b mAb was used as control. Pictures are representative of the results obtained with three RA patients and two patients with recurrent dislocation. (B and C) Immunofluorescence in RA synovium using a biotinylated anti-IL-26 mAb (red) and antibodies directed either against the cell lineage markers (green) CD68 and synoviolin (B), CD3 or RORgamma (C); bars = 100 µm. Pictures are representative of the results obtained with tissues from three RA patients.

Article Snippet: After saturation with PBS containing 2% non-fat dry milk, plates were successively incubated with samples or recombinant human IL-26, with a biotinylated polyclonal goat anti-IL-26 Ab and then with streptavidin-HRP (BD Pharmingen).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence

Journal: PLoS Biology

Article Title: IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

doi: 10.1371/journal.pbio.1001395

Figure Lengend Snippet: (A) FLS from RA patients and healthy subjects ( n = 3), and T cells and non-T mononuclear cells (isolated from RA SF cells and from PBMC of healthy subjects) ( n = 3), were either unstimulated or stimulated with anti-CD3 plus -CD28 mAbs or soluble CD40L, respectively. IL-26 mRNA expression (expressed as relative mRNA expression) was analyzed by RT-qPCR after 6 h stimulation (left panel) and IL-26 production (in ng/ml) was determined by ELISA after 48 h stimulation (right panel). Results are expressed as mean ± SD, n = 3; * p <0.05 (Wilcoxon matched-pairs signed-ranks test). (B) IL-26 (upper panel) and IL-6 (lower panel) were quantified by ELISA in the supernatants of FLS from healthy subjects and RA patients, stimulated or not with 50 ng/ml IL-1-beta and/or IL-17A. Results are expressed in ng/ml (mean ± SD, n = 3).

Article Snippet: After saturation with PBS containing 2% non-fat dry milk, plates were successively incubated with samples or recombinant human IL-26, with a biotinylated polyclonal goat anti-IL-26 Ab and then with streptavidin-HRP (BD Pharmingen).

Techniques: Isolation, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: PLoS Biology

Article Title: IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

doi: 10.1371/journal.pbio.1001395

Figure Lengend Snippet: (A) IL-1-beta, IL-6, and TNF-alpha were quantified by ELISA in the 16 h (IL-6 and TNF-alpha) or 48 h (IL-1-beta) supernatants of monocytes isolated from the blood of healthy subjects or CD14 + myeloid cells isolated from RA SF cells and exposed or not to 50 ng/ml IL-26. (B) Monocytes were exposed to increasing concentrations of IL-26. IL-1-beta, IL-6, and TNF-alpha were quantified by ELISA in the 16 h (IL-6 and TNF-alpha) or 48 h (IL-1-beta) supernatants. (C) Monocytes were exposed to 50 ng/ml IL-26 and IL-1-beta, IL-6, and TNF-alpha were quantified by ELISA in the supernatants collected at the indicated time-points. (D) Monocytes were exposed to 50 ng/ml IL-26 for 6 h and the expression of the mRNA encoding the indicated cytokines was analyzed by RT-qPCR. Results are expressed in fold increase of mRNA expression compared to unstimulated cells (mean ± SD, n = 3); nd, means not detectable. TNF-alpha mRNA expression, measured after 2 h stimulation , was increased of 15 fold (unpublished data). (E) Monocyte-derived macrophages (MΦ), BDCA1 + peripheral blood myeloid dendritic cells (mDC), and monocyte-derived DC (Mo-DC) were cultured without or with 50 ng/ml dimeric IL-26. IL-6 was quantified by ELISA in the 16 h supernatants. (F–G), Monocytes were exposed for 6 h to 50 ng/ml dimeric IL-26 (dim IL-26) and the mRNA encoding members of the IL-10 cytokine family (F) and chemokines (G) were analyzed by RT-qPCR. Results are expressed in fold increase of mRNA expression, compared to unstimulated cells. (H) Monocytes were cultured in the presence of 50 ng/ml IL-26, 50 ng/ml IL-26 plus 10 µg/ml neutralizing goat anti-IL-26 Ab or a control Ab, 50 ng/ml heat-treated IL-26, or 50 ng/ml monomeric IL-26 (mono IL-26). Insert, Western-blotting analysis of monomeric and dimeric IL-26. (I) Monocytes were cultured in the presence of 50 or 100 ng/ml IL-26 or 100 pg/ml LPS, with or without 0.2 µg/ml polymixin B. (J) Monocytes were cultured in the presence or absence of 50 ng/ml IL-26, with or without 20 ng/ml IL-4, IL-10, IL-13, TGF-beta, or TSLP. (H–J) IL-6 was quantified in the 16 h supernatants. (A–C, E, H–J) Results are expressed in ng/ml (mean ± SD, n = 5). * p <0.05 (Wilcoxon matched-pairs signed-ranks test).

Article Snippet: After saturation with PBS containing 2% non-fat dry milk, plates were successively incubated with samples or recombinant human IL-26, with a biotinylated polyclonal goat anti-IL-26 Ab and then with streptavidin-HRP (BD Pharmingen).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Quantitative RT-PCR, Derivative Assay, Cell Culture, Western Blot

Journal: PLoS Biology

Article Title: IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

doi: 10.1371/journal.pbio.1001395

Figure Lengend Snippet: (A) Naive or memory CD4 + T cells were stimulated by an anti-CD3 mAb, in the presence of monocytes, with or without 50 ng/ml IL-26 or 5 µg/ml PGN. IL-17A was quantified by ELISA in the 7-d supernatants. (B) Memory CD4 + T cells were stimulated by an anti-CD3 mAb, in the presence of monocytes, with or without IL-26 at the indicated concentrations. IL-17A was quantified in the 7-d supernatants. (C) Memory CD4 + T cells were stimulated as described, with or without 50 ng/ml IL-26. IFN-gamma, IL-4, IL-10, IL-21, and IL-22 were quantified by ELISA in the 7-d supernatants. (A–C) Results are expressed in ng/ml or pg/ml (mean ± SD, n = 5). (D and E) Memory CD4 + T cells, stimulated for 7 d by an anti-CD3 mAb in the presence of monocytes, with or without 50 ng/ml IL-26, were further cultured for 7 d. Then, cells were stimulated by PMA plus ionomycin and intracellular expression of IL-17A, IL-22, and IFNγ was analyzed by flow cytometry. (D) Dot plots are representative of one out of five experiments; (E) Results are expressed as a percentage of Th17- (IL-17A + ), Th1- (IFN-gamma + IL-17A − ), or Th22- (IL-22 + IL-17A − ) expressing cells (mean ± SD, n = 4). (F) Memory CD4 + T cells were stimulated by an anti-CD3 mAb in the presence of monocytes, with or without 50 ng/ml IL-26. At day 7, T cells were purified and IL-17A and RORgamma t mRNA expression was analyzed by RT-qPCR. Results are expressed as a relative mRNA expression level (mean ± SD, n = 4). * p <0.05 (Wilcoxon matched-pairs signed-ranks test).

Article Snippet: After saturation with PBS containing 2% non-fat dry milk, plates were successively incubated with samples or recombinant human IL-26, with a biotinylated polyclonal goat anti-IL-26 Ab and then with streptavidin-HRP (BD Pharmingen).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Flow Cytometry, Purification, Quantitative RT-PCR

Journal: PLoS Biology

Article Title: IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

doi: 10.1371/journal.pbio.1001395

Figure Lengend Snippet: (A) Memory CD4 + T cells were stimulated with an anti-CD3 Ab, with or without 50 ng/ml IL-26, in the presence of (i) monocytes, (ii) monocytes cultured in separate chambers (Transwell inserts), or (iii) an anti-CD28 Ab. Memory CD4 + T cells were also cultured with monocytes in the absence of anti-CD3 or anti-CD28 Ab. PGN (5 µg/ml) or 10 ng/ml IL-1-beta plus 50 ng/ml IL-6 were used as positive controls. After 1 wk, IL-17A was quantified by ELISA in the supernatants. Results are expressed in ng/ml (mean ± SD, n = 4). (B) Memory CD4 + T cells were stimulated by an anti-CD3 Ab plus monocytes, with or without 50 ng/ml IL-26, in the presence or absence of 10 µg/ml neutralizing anti-IL-1-beta, anti-IL-6, anti-TNF-alpha, isotype control Abs, or 1 µg/ml soluble IL-1RA. IL-17A was quantified after 1 wk. Results are expressed as percentage of inhibition of IL-17A secretion (mean ± SD, n = 4). (A and B) * p <0.05 (Wilcoxon matched-pairs signed-ranks test).

Article Snippet: After saturation with PBS containing 2% non-fat dry milk, plates were successively incubated with samples or recombinant human IL-26, with a biotinylated polyclonal goat anti-IL-26 Ab and then with streptavidin-HRP (BD Pharmingen).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: PLoS Biology

Article Title: IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

doi: 10.1371/journal.pbio.1001395

Figure Lengend Snippet: (A and B) Memory CD4 + T cell populations enriched in IL-17A-producing cells (>50%) were stimulated with an anti-CD3 mAb, in the presence of monocytes, with or without 50 ng/ml IL-26 or 5 µg/ml PGN. The frequency (measured at day 7; A) and proliferation (measured at day 5; B) of IL17A + , IL-17A − IFN-gamma + , and IL-17A − IFN-gamma − T cells were analyzed by flow cytometry 6 h after stimulation with PMA plus ionomycin. Results are expressed as a percentage of variation of T cell frequency after stimulation, compared to untreated cells (A) or as a percentage of proliferating T cells (B) (mean ± SD, n = 4). (C) FACS-sorted IL-23R − , CCR6 − CD161 − , and CCR6 + CD161 + memory CD4 + T cells were stimulated by an anti-CD3 mAb in the presence monocytes, with or without IL-26. IL-17A was quantified after 1 wk. Results are expressed in ng/ml (mean ± SD, n = 4).

Article Snippet: After saturation with PBS containing 2% non-fat dry milk, plates were successively incubated with samples or recombinant human IL-26, with a biotinylated polyclonal goat anti-IL-26 Ab and then with streptavidin-HRP (BD Pharmingen).

Techniques: Flow Cytometry

Journal: PLoS Biology

Article Title: IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

doi: 10.1371/journal.pbio.1001395

Figure Lengend Snippet: (A and B) IL-1-beta (A) and IL-6 (B) were quantified by ELISA in the 48 h supernatants of monocytes (isolated from healthy subjects) cultured in X-VIVO-20 medium, supplemented with 10% human serum of three RA patients (which contained 10, 12, and 17 ng/ml of IL-26) or with 10% SFs of two RA patients (which contained 80 and 68 ng/ml of IL-26), either depleted or not in IL-26. Culture medium supplemented by 10% serum of RA patients or of healthy subjects contained less than 12 pg/ml IL-6, and IL-1-beta was undetectable in the culture medium supplemented by 10% SF and serums of RA patients. Experiments were performed with monocytes from four healthy subjects and results are expressed in ng/ml (mean ± SD, n = 4). For healthy subjects, the result is representative of one of four serums. (C) Memory CD4 + T cells were stimulated with an anti-CD3 mAb in the presence of monocytes, supplemented by 10% SF of three RA patients, depleted or not in IL-26, or with 10% serum of three healthy subjects, used as a control. IL-17A was quantified by ELISA in the 7-d supernatants. Results are expressed in ng/ml (mean ± SD, n = 3), and are representative of the results obtained with monocytes and T cells from one out of three healthy subjects. * p <0.05 (Wilcoxon matched-pairs signed-ranks test).

Article Snippet: After saturation with PBS containing 2% non-fat dry milk, plates were successively incubated with samples or recombinant human IL-26, with a biotinylated polyclonal goat anti-IL-26 Ab and then with streptavidin-HRP (BD Pharmingen).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Cell Culture

Journal: PLoS Biology

Article Title: IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

doi: 10.1371/journal.pbio.1001395

Figure Lengend Snippet: In inflamed RA joints, IL-26 is constitutively produced by synoviocytes and upregulates IL-1-beta and CCL20 secretion by myeloid cells. IL-1-beta favors the polarization of infiltrating CD4 + memory T cells into Th17 cells, and CCL20 promotes the recruitment of CCR6 + Th17 cells. In parallel, IL-1-beta and IL-17A, produced by monocytes and Th17 cells, respectively, increase IL-26 secretion by synoviocytes. IL-26 secreted by Th17 cells , may also contribute to the production of proinflammatory cytokines by monocytes. Moreover, CCL20 produced by activated FLS may also contribute to local inflammation by inducing the recruitment of pathogenic Th17 cells in the synovium , . This scheme was drawn using pictures from Servier Medical Art.

Article Snippet: After saturation with PBS containing 2% non-fat dry milk, plates were successively incubated with samples or recombinant human IL-26, with a biotinylated polyclonal goat anti-IL-26 Ab and then with streptavidin-HRP (BD Pharmingen).

Techniques: Produced