Journal: Frontiers in Microbiology

Article Title: IL-36γ Augments Host Defense and Immune Responses in Human Female Reproductive Tract Epithelial Cells

doi: 10.3389/fmicb.2016.00955

Figure Lengend Snippet: Human 3-D vaginal and cervical epithelial cells and tissue express IL-36γ and IL-36R. (A) Expression of IL-36γ and IL-36R in 3-D human vaginal and endocervical cell cultures, and human vaginal and cervical tissue. cDNA from cell cultures and tissue were analyzed by qRT-PCR and mRNA copy numbers were normalized against GAPDH. (B) Cell lysates from 3-D vaginal and endocervical cell cultures, vaginal tissue and cervical tissue were evaluated by Western blot for expression of IL-36γ. β-tubulin was used as a loading control. 30 μg total protein from each lysate was loaded. (C) TNFα (100 ng/ml) treated 3-D cultures of vaginal and endocervical epithelial cells expressed IL-36γ. mRNA levels were normalized against GAPDH and expression of IL-36γ was reported as fold change compared to PBS treated samples. Data are shown as mean ± SD from three independent experiments each performed in duplicate. ˆ P < 0.05; ∗ P < 0.01; ∗∗ P < 0.001; unpaired two-tailed Student t -test with Welch’s correction.

Article Snippet: Both 3-D vaginal EC and 3-D endocervical EC were treated with poly (I:C; InvivoGen) at 1, 25, and 100 μg/ml, flagellin (InvivoGen) at 0.5 and 5 μg/ml, FSL-1 (InvivoGen) at 0.01 and 0.1 μg/ml, or recombinant IL-36γ (PeproTech, Rocky Hill, NJ, USA) at 1, 10, 100, and 500 ng/ml for 24 h at 37°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: IL-36γ Augments Host Defense and Immune Responses in Human Female Reproductive Tract Epithelial Cells

doi: 10.3389/fmicb.2016.00955

Figure Lengend Snippet: Differential IL-36γ induction in 3-D human vaginal and endocervical epithelial cells treated with microbial products. Gene expression analysis of 3-D vaginal and endocervical EC. Cell cultures were exposed to microbial products [poly (I:C); 1, 25, and 100 μg/ml, flagellin (FLA); 0.5 and 5 μg/ml, and fibroblast stimulating lipopeptide-1 (FSL-1); 0.01 and 0.1 μg/ml] for 24 h. Expression of IL-36γ was determined by qRT-PCR and reported relative to GAPDH as fold change compared to PBS treated samples. Horizontal dashed line indicates IL-36γ expression in PBS treated cells. Data are shown as mean ± SD from three independent experiments each performed in duplicate. ˆ P < 0.05; ∗ P < 0.01; ∗∗ P < 0.001; unpaired two-tailed Student t -test with Welch’s correction.

Article Snippet: Both 3-D vaginal EC and 3-D endocervical EC were treated with poly (I:C; InvivoGen) at 1, 25, and 100 μg/ml, flagellin (InvivoGen) at 0.5 and 5 μg/ml, FSL-1 (InvivoGen) at 0.01 and 0.1 μg/ml, or recombinant IL-36γ (PeproTech, Rocky Hill, NJ, USA) at 1, 10, 100, and 500 ng/ml for 24 h at 37°C.

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: IL-36γ Augments Host Defense and Immune Responses in Human Female Reproductive Tract Epithelial Cells

doi: 10.3389/fmicb.2016.00955

Figure Lengend Snippet: Extracellular and intracellular localization of IL-36γ in epithelial cells of the human female reproductive tract following treatment with microbial products. Three-D vaginal and endocervical EC were treated with PBS, flagellin (FLA; 5 or 0.5 μg ml -1 ), FSL-1 (0.1 or 0.01 μg ml -1 ), or poly (I:C; 100, 25, or 1 μg ml -1 ) for 24 h. (A) Cell culture lysates and (B) supernatants were assayed by ELISA to quantify IL-36γ production and secretion. Levels of IL-36γ were determined and reported as fold change relative to PBS treated samples. Horizontal dashed line indicates IL-36γ expression in PBS treated cells. Data are shown as mean ± SD from three independent experiments each performed in duplicate. ˆ P < 0.05; ∗ P < 0.01; ∗∗ P < 0.001; unpaired 2-tailed Student t -test with Welch’s correction.

Article Snippet: Both 3-D vaginal EC and 3-D endocervical EC were treated with poly (I:C; InvivoGen) at 1, 25, and 100 μg/ml, flagellin (InvivoGen) at 0.5 and 5 μg/ml, FSL-1 (InvivoGen) at 0.01 and 0.1 μg/ml, or recombinant IL-36γ (PeproTech, Rocky Hill, NJ, USA) at 1, 10, 100, and 500 ng/ml for 24 h at 37°C.

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

Journal: Frontiers in Microbiology

Article Title: IL-36γ Augments Host Defense and Immune Responses in Human Female Reproductive Tract Epithelial Cells

doi: 10.3389/fmicb.2016.00955

Figure Lengend Snippet: Induction of IL-36γ and IL-36R expression following recombinant IL-36γ treatment. 3-D vaginal and endocervical EC were treated with increasing doses of recombinant IL-36γ (1, 10, 100, and 500 ng/ml) for 24 h. (A) IL-36γ and (B) IL-36R expression was determined by qRT-PCR and reported relative to GAPDH as fold change compared to PBS treated samples. Horizontal dashed line indicates IL-36γ expression in PBS treated cells. Data are shown as mean ± SD from three independent experiments each performed in duplicate. ˆ P < 0.05; ∗ P < 0.01; ∗∗ P < 0.001; unpaired two-tailed Student t -test with Welch’s correction.

Article Snippet: Both 3-D vaginal EC and 3-D endocervical EC were treated with poly (I:C; InvivoGen) at 1, 25, and 100 μg/ml, flagellin (InvivoGen) at 0.5 and 5 μg/ml, FSL-1 (InvivoGen) at 0.01 and 0.1 μg/ml, or recombinant IL-36γ (PeproTech, Rocky Hill, NJ, USA) at 1, 10, 100, and 500 ng/ml for 24 h at 37°C.

Techniques: Expressing, Recombinant, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: IL-36γ Augments Host Defense and Immune Responses in Human Female Reproductive Tract Epithelial Cells

doi: 10.3389/fmicb.2016.00955

Figure Lengend Snippet: Recombinant IL-36γ treatment induces antimicrobial peptide expression in 3-D human vaginal and endocervical epithelial cells. 3-D vaginal and endocervical cell cultures were treated with increasing doses of recombinant IL-36γ (1, 10, 100, or 500 ng/ml) for 24 h. Relative expression levels of (A) IL-8, (B) CCL20, (C) HE4, (D) SLPI, (E) HBD-2, and (F) HBD-4 were determined by qRT-PCR and reported relative to GAPDH as fold change compared to PBS treated cells Data are shown as mean ± SD from three independent experiments each performed in duplicate. ˆ P < 0.05; ∗ P < 0.01; ∗∗ P < 0.001; unpaired two-tailed Student t -test with Welch’s correction.

Article Snippet: Both 3-D vaginal EC and 3-D endocervical EC were treated with poly (I:C; InvivoGen) at 1, 25, and 100 μg/ml, flagellin (InvivoGen) at 0.5 and 5 μg/ml, FSL-1 (InvivoGen) at 0.01 and 0.1 μg/ml, or recombinant IL-36γ (PeproTech, Rocky Hill, NJ, USA) at 1, 10, 100, and 500 ng/ml for 24 h at 37°C.

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: IL-36γ Augments Host Defense and Immune Responses in Human Female Reproductive Tract Epithelial Cells

doi: 10.3389/fmicb.2016.00955

Figure Lengend Snippet: 3-D human vaginal and endocervical epithelial cells secrete increased levels of pro-inflammatory cytokines and chemokines in response to treatment with recombinant IL-36γ. 3-D vaginal and endocervical cell cultures were treated with PBS or increasing doses of recombinant IL-36γ (1, 10, 100, or 500 ng/ml) for 24 h. Expression levels of (A) IL-1β, (B) IL-6, (C) TNFα, and chemokine (D) CCL20 were determined by multiplex analysis and reported in pg/ml. Data are shown as mean ± SD from three independent experiments each performed in duplicate. ˆ P < 0.05; ∗ P < 0.01; ∗∗ P < 0.001; unpaired two-tailed Student t -test with Welch’s correction.

Article Snippet: Both 3-D vaginal EC and 3-D endocervical EC were treated with poly (I:C; InvivoGen) at 1, 25, and 100 μg/ml, flagellin (InvivoGen) at 0.5 and 5 μg/ml, FSL-1 (InvivoGen) at 0.01 and 0.1 μg/ml, or recombinant IL-36γ (PeproTech, Rocky Hill, NJ, USA) at 1, 10, 100, and 500 ng/ml for 24 h at 37°C.

Techniques: Recombinant, Expressing, Multiplex Assay, Two Tailed Test

Journal: iScience

Article Title: Immunometabolic profiling of cervicovaginal lavages identifies key signatures associated with adenomyosis

doi: 10.1016/j.isci.2022.105508

Figure Lengend Snippet:

Article Snippet: Levels of IL-36γ (IL-1F9) were measured in the samples by enzyme-linked immunosorbent assay using Human IL-36γ ELISA kit (RayBiotech, Norcross, GA) in accordance with the manufacturer’s instructions.

Techniques: Recombinant, Biomarker Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: PLoS ONE

Article Title: Interleukin (IL)-1β Is a Strong Inducer of IL-36γ Expression in Human Colonic Myofibroblasts

doi: 10.1371/journal.pone.0138423

Figure Lengend Snippet: (A) IL-36γ mRNA expression in human colonic myofibroblasts. The cells were stimulated with cytokines (100 ng/ml) for 24h, and IL- 36γ mRNA expression was analyzed by real-time PCR. IL-36γ mRNA expression was expressed as relative to β-actin mRNA expression (mean ± SD from 4 different experiments). **P < 0.01 versus medium only. (B) IL-36γ protein expression. The cells were stimulated with or without IL-1β (10 ng/ml) for 24h, and intracellular IL-36γ was analyzed by Western blot.

Article Snippet: IL-36γ protein in the cell culture supernatant was quantified by an ELISA kit, purchased from Uscn Life Science (Wuhan, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Interleukin (IL)-1β Is a Strong Inducer of IL-36γ Expression in Human Colonic Myofibroblasts

doi: 10.1371/journal.pone.0138423

Figure Lengend Snippet: (A) Dose-dependent effects of IL-1β on IL-36γ mRNA expression. The cells were incubated for 24 h with increasing concentrations of IL-1β. IL-36γ mRNA expression was expressed relative to β-actin mRNA expression (mean ± SD from 4 different experiments). (B) Time-dependent effects of IL-1β on IL-36γ mRNA expression. The cells were stimulated with IL-1β (10 ng/ml) for the pre-determined times. IL-36γ mRNA expression was expressed relative to β-actin mRNA expression (mean ± SD from 4 different experiments). (C) Dose-dependent effects of IL-1β on IL-36γ secretion. The cells were incubated for 24 h with increasing concentrations of IL-1β. IL-36γ level in supernatant was determined by ELISA (mean ± SD from 4 different experiments). (D) Time-dependent effects of IL-1β on IL-36γ secretion. The cells were stimulated with IL-1β (10 ng/ml) for the pre-determined times. IL-36γ level was determined by ELISA (mean ± SD from 4 different experiments). *P<0.05, **P<0.01 versus medium; ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: IL-36γ protein in the cell culture supernatant was quantified by an ELISA kit, purchased from Uscn Life Science (Wuhan, China).

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Interleukin (IL)-1β Is a Strong Inducer of IL-36γ Expression in Human Colonic Myofibroblasts

doi: 10.1371/journal.pone.0138423

Figure Lengend Snippet: (A) The cells were incubated for 24 h with combination of IL-1β (10 ng/ml) and other cytokines [TNF-α (100 ng/ml), IFN-γ (100 ng/ml), IL-4 (100 ng/ml), and/or IL-17A (100 ng/ml)]. IL-36γ mRNA expression was analyzed by real-time PCR. IL-36γ mRNA expression was expressed relative to the β-actin mRNA expression (mean ± SD from 4 different experiments). **P<0.01 versus IL-1β alone. (B) The cells were stimulated for 24 h with or without IL-1β (10 ng/ml), TNF-α (100 ng/ml), and/or combination of IL-1β (10 ng/ml) and TNF-α (100 ng/ml), and intracellular IL-36γ was analyzed by Western blot.

Article Snippet: IL-36γ protein in the cell culture supernatant was quantified by an ELISA kit, purchased from Uscn Life Science (Wuhan, China).

Techniques: Incubation, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Interleukin (IL)-1β Is a Strong Inducer of IL-36γ Expression in Human Colonic Myofibroblasts

doi: 10.1371/journal.pone.0138423

Figure Lengend Snippet: (A) MAPK activation in colonic SEMFs. The cells were stimulated with IL-1β (10 ng/ml), and the activation of MAPKs were analyzed by Western blotting. Antibodies against phosphorylated (p)- and total- MAPKs were used. *P < 0.05, **P<0.01. (B) The cells were stimulated for 24 h with IL-1β (10ng/ml) in the presence or absence of MEK inhibitors [U0216 (10 μM) and PD98059 (10 μM)] and a p38 inhibitor [SB203580 (10 μM)]. IL-36γ mRNA expression was analyzed by real-time PCR. IL-36γ mRNA expression was expressed relative to the β-actin mRNA expression (mean ± SD from 4 different experiments).

Article Snippet: IL-36γ protein in the cell culture supernatant was quantified by an ELISA kit, purchased from Uscn Life Science (Wuhan, China).

Techniques: Activation Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Interleukin (IL)-1β Is a Strong Inducer of IL-36γ Expression in Human Colonic Myofibroblasts

doi: 10.1371/journal.pone.0138423

Figure Lengend Snippet: (A) IL-1β induced IκBα phosphorylation and degradation in colonic myofibroblasts. The cells were stimulated with IL-1β (10 ng/ml), and phosphorylated IκBα were detected by Western blotting. (B) IL-1β induced activation of NF-κB and c-Jun AP-1. The cells were stimulated with IL-1β (10 ng/ml), and NF-κB p65 and phosphorylated c-Jun were detected by immunocytochemistory. Reacted antibodies against NF-κB p65 were visualized by FITC (green fluorescence)-labeled second antibody. Reacted antibodies against phosphorylated c-Jun were detected by a DyLight ® 594 (red fluorescence)-labeled secondary antibodies. Nucleus was stained by DAPI (blue). (C) The cells were stimulated with IL-1β (10 ng/ml) or medium alone for 15 min and nuclear proteins were extracted. NF-κB p65 and phosphorylated c-Jun in nuclear extracts were detected by immunoblot. (D) Effects of silencing of NF-κB p65 and c-Jun AP-1on IL-1β-induced IL-36γ expression. The cells were transfected with control siRNA, the siRNA specific for NF-κB p65 and/or c-Jun AP-1, and incubated for 24h. IL-36γ mRNA expression was analyzed by real-time PCR. IL-36γ mRNA expression was expressed relative to the β-actin mRNA expression (mean ± SD from 4 different experiments). *P<0.05, **P < 0.01 versus IL-1β stimulation.

Article Snippet: IL-36γ protein in the cell culture supernatant was quantified by an ELISA kit, purchased from Uscn Life Science (Wuhan, China).

Techniques: Western Blot, Activation Assay, Fluorescence, Labeling, Staining, Expressing, Transfection, Incubation, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: Inflammation modulates intercellular adhesion and mechanotransduction in human epidermis via ROCK2

doi: 10.1016/j.isci.2023.106195

Figure Lengend Snippet:

Article Snippet: IL-36γ reverse primer 5′-GGT CCA CAC TTG CTG ATT CAA A-3′ , Microsynth , N/A.

Techniques: Recombinant, Permeability, Gene Expression, Software

Journal: The Journal of investigative dermatology

Article Title: Unprocessed interleukin-36α regulates psoriasis-like skin inflammation in co-operation with interleukin-1

doi: 10.1038/jid.2015.289

Figure Lengend Snippet: Wild type, IL-36α −/− , IL-36β −/− and IL-36γ −/− mice were treated with imiquimod for 4 days. The day after the last application anesthetized ( a ) or euthanized ( b ) mice were photographed and skin collected after euthanasia. Control mice were denuded and left untreated until skin collection. Skin was examined by H&E staining ( c–d ) and immunohistochemistry for PCNA ( e–f ). Representative images from 5 (IL-36α −/− and IL-36β −/− ) and 3 (IL-36γ −/− ) independent imiquimod experiments are shown. Blue scale bars = 200 µm. Black scale bars = 50 µm.

Article Snippet: Proteins were separated by PAGE, transferred to PVDF membranes and detected by ECL using anti-IL-36α (AF2297), anti-IL-36γ (custom synthesized at Genscript) or anti-GAPDH (FL-335, Santa Cruz) and appropriate secondary antibodies.

Techniques: Control, Staining, Immunohistochemistry

Journal: The Journal of investigative dermatology

Article Title: Unprocessed interleukin-36α regulates psoriasis-like skin inflammation in co-operation with interleukin-1

doi: 10.1038/jid.2015.289

Figure Lengend Snippet: Wild type ( a and b ), IL-36α −/− ( a ), IL-36β −/− ( a ) and IL-36γ −/− ( b ) mice were treated with imiquimod for 4 days as described in . Skin was collected the day after the last imiquimod application and examined by H&E staining or immunohistochemistry for neutrophils (Ly-6G/Ly-6C). Red arrows indicate Munro’s microabscesses (clusters of neutrophils) at the top of the epidermis. Black arrows indicate individual neutrophils within the dermis. Representative images from the experiments also analyzed in and are shown. Scale bars = 50 µm.

Article Snippet: Proteins were separated by PAGE, transferred to PVDF membranes and detected by ECL using anti-IL-36α (AF2297), anti-IL-36γ (custom synthesized at Genscript) or anti-GAPDH (FL-335, Santa Cruz) and appropriate secondary antibodies.

Techniques: Staining, Immunohistochemistry