Journal: Frontiers in immunology
Article Title: A Reappraisal on the Potential Ability of Human Neutrophils to Express and Produce IL-17 Family Members In Vitro : Failure to Reproducibly Detect It.
doi: 10.3389/fimmu.2018.00795
Figure Lengend Snippet: Figure 2 | No induction of IL-17A, IL-17F, and IL-17RC mRNA expression in neutrophils incubated with IL-6 plus IL-23, in combination with inactivated Aspergillus fumigatus hyphae or conidia. Neutrophils (5 × 106/ml) were incubated either with 100 ng/ml rIL-17A for 2 h or with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23 for 1 h, prior to adding, or not, inactivated A. fumigatus conidia (1:5 neutrophils/conidia ratio) and hyphae (1:1 neutrophils/hyphae ratio) for additional 1 h. Neutrophils were then harvested for RNA extraction to evaluate IL-17A (A), IL-17F (B), IL-17RC (C), IL-17RA (D), and SOCS3 (F) mRNA expression by reverse transcription quantitative real-time PCR. Gene expression data are depicted as mean normalized expression (MNE) units after GAPDH mRNA normalization (mean ± SEM, n = 4). Asterisks stand for significant differences as compared to untreated cells: *P < 0.05, **P < 0.01, by Student’s t-test. (E) Immunoblot displaying STAT3 tyrosine phosphorylation in neutrophils, either untreated or cultured for 15 or 60 min with 20 µg/ml IL-6 plus 2 µg/ml IL-23 (representative experiment, n = 2).
Article Snippet: Protein-rich flow-through from neutrophils were then immunoblotted by standard procedures using the anti-human IL-17A (AF-317-NA) and IL-17B (AF1248) goat IgG pAbs from R&D Systems; anti-human phospho-STAT3 (Tyr705) rabbit pAbs (#9131, Cell Signaling, Beverly, MA, USA); anti-human STAT3 rabbit pAbs (sc-482, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-human β-actin mAbs (A5060 from Sigma).
Techniques: Expressing, Incubation, RNA Extraction, Reverse Transcription, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Phospho-proteomics, Cell Culture
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![Figure 8 | Staining human neutrophils by anti-IL-17A (AF-317-NA) polyclonal antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) stainings of two FFPE cases of human pustular psoriasis using anti-IL-17A (AF-317-NA) and anti-CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A+ cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils, either untreated (top panels) or treated with 5 µM R848 (bottom panels) for 3 h, were stained with anti-IL-17A (AF-317-NA, left panels) and anti-CXCL8 (right panels) Abs. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A), center/right images in third row in (A), as well as in (B), scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF-317-NA immunoblot of lysates from neutrophils either freshly isolated (T0, from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17A (rhIL-17A) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_9541/pm29719541/pm29719541__page13_image1.jpg)
![Figure 10 | Staining human neutrophils by anti-IL-17B (AF1248) antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) staining of two FFPE cases of human pustular psoriasis using anti-IL-17B (AF1248) and CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A+ cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils incubated without (top panel) or with 5 µM R848 (bottom panel) for 3 h. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A), center/right images in third row in (A), as well as in (B), scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF1248 immunoblot of lysates from neutrophils either freshly isolated (T0, from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17B (rhIL-17B) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_9541/pm29719541/pm29719541__page15_image1.jpg)