For the cytokine studies, the media was changed every 2 days. For the IL‐13 experiments, 20 ng/ml IL‐13 (R&D Systems) or DMSO vehicle was added to the basolateral compartment of differentiated epithelia, then 20 µl of the basolateral solution was added to the apical surface and experiments were performed 21 days after initial treatment. 20 ng/ml IL‐13 is sufficient to increase goblet cell abundance in many laboratories (Laoukili et al. 2001 ; Atherton et al. 2003 ; Zhen et al. 2007 ; Kanoh et al. 2011 ; Thavagnanam et al. 2011 ; Dickinson et al. 2016 ; Pezzulo et al. 2019 ). For IL‐17/TNFα experiments, 20 ng/ml IL‐17 (R&D Systems) and 10 ng/ml TNFα (R&D Systems) or DMSO was added to the basolateral media and experiments were performed 2 days later based on preliminary dose–response studies and previous reports (Kao et al. 2004 ; McAllister et al. 2005 ; Kreindler et al. 2009 ; Choy et al. 2015 ; Lehmann et al. 2018 ; Pezzulo et al. 2019 ).
Il 17
Manufactured by R&D Systems
Sourced in United States, United Kingdom, Germany
Interleukin-17 (IL-17) is a cytokine that plays a crucial role in inflammatory and autoimmune responses. It is predominantly produced by a specialized subset of T cells known as Th17 cells. IL-17 functions to induce the expression of other inflammatory mediators, thereby contributing to the recruitment and activation of immune cells at sites of inflammation.
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Lab products found in correlation
Tnf α, by R&D Systems (45 mentions)
Interleukin 6 (il 6), by R&D Systems (36 mentions)
Il 1β, by R&D Systems (31 mentions)
Ifn γ, by R&D Systems (26 mentions)
Interleukin 4 (il 4), by R&D Systems (14 mentions)
Il 22, by R&D Systems (13 mentions)
Il 10, by R&D Systems (12 mentions)
Il 23, by R&D Systems (12 mentions)
Ifn γ, by BD (10 mentions)
Tgf β, by R&D Systems (8 mentions)
Il 13, by R&D Systems (8 mentions)
Sytox green, by Thermo Fisher Scientific (8 mentions)
Tgf β1, by R&D Systems (6 mentions)
Gm csf, by R&D Systems (6 mentions)
Ifn γ, by Thermo Fisher Scientific (6 mentions)
Photomicroscope, by Olympus (5 mentions)
Tumor necrosis factor (tnf), by R&D Systems (5 mentions)
Il 10, by BD (5 mentions)
Interleukin 4 (il 4), by BD (5 mentions)
Tnf α, by BD (5 mentions)
154 protocols using il 17
1
Cytokine-induced Goblet Cell Differentiation
2
Neutrophil and CD8+ T Cell Migration Assays
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Migration of neutrophils, isolated from PBMC of HDs by magnetic beads (EasyStep enrichment kit, StemCell Technologies), was assessed in transwell plates (5 µm pore size, Corning Costar), towards Th17 supernatants or recombinant cytokines (IL-17, R&D Systems, 50 ng/mL; IL-8, R&D Systems, 100 ng/mL; GM-CSF, R&D Systems, 100 ng/mL), for 90 min at 37°C. In specific experiments, anti-IL-8 or anti-GM-CSF antibodies (10 μg/mL, R&D Systems) were added to Th17 supernatants.
Migration of CD8+ T cells, sorted from PBMC of HDs by magnetic microbeads (Miltenyi Biotec) and pre-activated overnight with anti-CD3/CD28 antibodies, was evaluated in transwell plates (5 µm pore size) towards Th17 supernatants, supernatants of HMEC cells, untreated, or exposed overnight to Th17 supernatants or recombinant proteins (IL-17, 50 ng/mL; TNF-α, 1 ng/mL, R&D Systems; IFN-γ, 1 ng/mL, Biolegend; or their combination), or towards CCL5 (60 and 200 ng/mL, R&D Systems) and/or CCL20 (300 and 1000 ng/mL, R&D Systems). Depletion of CCL5 and/or CCL20 from Th17-derived supernatants was obtained by specific capture antibodies (R&D Systems). Cell migration was quantified by flow cytometry.
Migration of CD8+ T cells, sorted from PBMC of HDs by magnetic microbeads (Miltenyi Biotec) and pre-activated overnight with anti-CD3/CD28 antibodies, was evaluated in transwell plates (5 µm pore size) towards Th17 supernatants, supernatants of HMEC cells, untreated, or exposed overnight to Th17 supernatants or recombinant proteins (IL-17, 50 ng/mL; TNF-α, 1 ng/mL, R&D Systems; IFN-γ, 1 ng/mL, Biolegend; or their combination), or towards CCL5 (60 and 200 ng/mL, R&D Systems) and/or CCL20 (300 and 1000 ng/mL, R&D Systems). Depletion of CCL5 and/or CCL20 from Th17-derived supernatants was obtained by specific capture antibodies (R&D Systems). Cell migration was quantified by flow cytometry.
3
IL-17 Stimulation and Blocking Assay
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MSCs, MC3T3‐E1, MLO‐A5, and MLO‐Y4 cells were stimulated with 0.5, 5, or 50 ng/ml IL‐17 (R&D Systems, Minneapolis, MN) diluted in the medium. For the blocking assay, cells were treated with 0.8 μg/ml IL‐17A receptor neutralizing antibody (R&D Systems) for 2 hr in serum‐free medium, and then stimulated with 50 ng/ml IL‐17. The control group was cultured in the standard medium under the same conditions.
4
Quantification of Immune Mediators
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Ear tissues were homogenized and collected into a protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA). After vortexing, the homogenates were centrifuged and the clear supernatants collected. IL-4, IL-6, IL-17, IFN-γ, TGF-β1, and TNF-α (R&D Systems, Minneapolis, MN, USA), IL-22 and IgE (Biolegend, San Diego, CA, USA) in tissue supernatants or plasma were measured by enzyme-linked immunosorbent assays (ELISA) according to the manufacturers’ instructions.
5
Protein Quantification in Lung Tissues
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Each lung tissue specimen was homogenized using cell lysis buffer. The cell lysis buffer consisting of 50 mM Tris-HCl pH 7.2, 0.5 mM EDTA, 0.15 M NaCl, and 0.5% NP-40. The homogenates were then centrifuged at 100,000× g for 1 h. Total protein concentrations in the supernatants were measured using the Bio-Rad Protein Assay to normalize the concentration of target cytokines in the supernatants. In the manner described above for BALF, tissue supernatant was assayed for the protein levels of TNF-α, GM-CSF, IL-17, and IL-33 (R&D Systems, Minneapolis, MN, USA).
6
Modulating YAP Signaling in HEK293 Cells
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HEK293 cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO, US) with 10% fetal bovine serum (FBS), 1% non-essential amino acid solution and 2% penicillin and streptomycin (PS). HEK293 YAP−/− were generated using specific CRISPR cas-9 and homology direct repair plasmid targeting YAP sequence (Santa Cruz Biotechnology, Dallas, TX, US), CRISPR clones generation was done following manufacturer instructions, and validated by western blot and DNA sequencing. Plates were coated with fibronectin (1:100, Sigma) for 2 h at 37°C before seeding. HEK293 were plated at high cell density (100,000 cells/cm2) or low cell density (10,000 cells/cm2). Approximately 24 h after seeding, TNF was used between 2.5 and 50 ng/ml (except if specified) and IL-17 at 50 ng/ml (R&D system, Minneapolis, MN, US. TNF or IL-17 treatment was performed for 48 h (except for luciferase assay). Y27632 (Sigma) was used at 10 µM and was pre-incubated for 2 h before TNF addition. C3 exoenzyme cell permeable (cytoskeleton, Denver, CO, US) was used at 2 µg/ml and pre-incubated for 2 h before TNF addition.
7
Intranasal IL-17 Induces Lung Inflammation
8
Transcriptional Profiling of IL-17 and TNF Stimulated Human Placental Cells
9
Spinal Cord Tissue RNA Extraction
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Rats were deeply anesthetized by inhalation of isoflurane (4%) in 100% O2 and intracardially perfused with 0.9% saline (between 200 and 250 ml per rat at 4°C), and the spinal cords were subsequently harvested. The lumbar spinal dorsal horns from the L5 to L6 ipsilateral to injury were dissected and rapidly frozen. The tissues were homogenized and total RNA was extracted with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). In the in vitro experiment, total RNA was extracted from astrocytes with 100 ng/ml IL-17 (R&D Systems, Inc., Minneapolis, MN, USA) treatment for three days or without. Interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and/or IL-17 mRNA were measured by RT-qPCR. RT-qPCR was performed with the Verso 1-step RT-qPCR kit, SYBR Green, low ROX (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and the thermocycling conditions were as follows: 5 min at 94°C; followed by 35 cycles of 10 sec at 94°C, 5 sec at 50°C, and 10 sec at 72°C. Relative expression was calculated with normalization to β-actin values (26 (link)). The primers used are presented in Table I .
10
ATDC5 Chondrocyte Modulation by IL-17 and Apremilast
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The ATDC5 chondrocyte cell line was obtained from the Cell Bank of the Cell Engineering Division, RIKEN BioResource Research Center. The cell line was maintained in DMEM/F12 medium (cat. no. 12634010) and supplied with 10% FBS (cat. no. 10100147; both from Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO2 humidified cell culture incubator at 37℃. For the treatment experiment, the cells were plated in 6-well cell culture plates and treated with IL-17 (10 ng/ml) (R&D Systems, Inc.) in the presence or absence of apremilast (0.5 and 1 µM) (MedChemExpress) at 37°C for 24 h.
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