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: FIGURE 1 | 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. ˆP < 0.05; ∗P < 0.01; ∗∗P < 0.001; unpaired two-tailed Student t-test with Welch’s correction.

Article Snippet: The microtiter plates were then washed three times with PBST then biotinylated polyclonal goat anti-human IL-36γ detection antibody (R&D Systems) diluted at 2 μg/ml with 1% skim milk in PBST was added to each well and incubated for 2 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: FIGURE 2 | 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. ˆP < 0.05; ∗P < 0.01; ∗∗P < 0.001; unpaired two-tailed Student t-test with Welch’s correction.

Article Snippet: The microtiter plates were then washed three times with PBST then biotinylated polyclonal goat anti-human IL-36γ detection antibody (R&D Systems) diluted at 2 μg/ml with 1% skim milk in PBST was added to each well and incubated for 2 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: FIGURE 3 | 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. ˆP < 0.05; ∗P < 0.01; ∗∗P < 0.001; unpaired 2-tailed Student t-test with Welch’s correction.

Article Snippet: The microtiter plates were then washed three times with PBST then biotinylated polyclonal goat anti-human IL-36γ detection antibody (R&D Systems) diluted at 2 μg/ml with 1% skim milk in PBST was added to each well and incubated for 2 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: FIGURE 4 | 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. ˆP < 0.05; ∗P < 0.01; ∗∗P < 0.001; unpaired two-tailed Student t-test with Welch’s correction.

Article Snippet: The microtiter plates were then washed three times with PBST then biotinylated polyclonal goat anti-human IL-36γ detection antibody (R&D Systems) diluted at 2 μg/ml with 1% skim milk in PBST was added to each well and incubated for 2 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: FIGURE 5 | 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. ˆP < 0.05; ∗P < 0.01; ∗∗P < 0.001; unpaired two-tailed Student t-test with Welch’s correction.

Article Snippet: The microtiter plates were then washed three times with PBST then biotinylated polyclonal goat anti-human IL-36γ detection antibody (R&D Systems) diluted at 2 μg/ml with 1% skim milk in PBST was added to each well and incubated for 2 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: FIGURE 6 | 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. ˆP < 0.05; ∗P < 0.01; ∗∗P < 0.001; unpaired two-tailed Student t-test with Welch’s correction.

Article Snippet: The microtiter plates were then washed three times with PBST then biotinylated polyclonal goat anti-human IL-36γ detection antibody (R&D Systems) diluted at 2 μg/ml with 1% skim milk in PBST was added to each well and incubated for 2 h at 37◦C.

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

Journal: Cell reports

Article Title: Chromatin Landscape Underpinning Human Dendritic Cell Heterogeneity

doi: 10.1016/j.celrep.2020.108180

Figure Lengend Snippet:

Article Snippet: All conditions included 10 ng/mL recombinant human IL-3 (R&D Systems; carrier-free) for pDC survival.

Techniques: Purification, Recombinant, Produced, SYBR Green Assay, Saline, Lysis, Electron Microscopy, Labeling, Staining, Cell Isolation, Enzyme-linked Immunosorbent Assay, Control, Antibody Labeling, Reverse Transcription, DNA Library Preparation, Generated, Microarray, Software

Journal: Immunology letters

Article Title: Assessment of faithful interleukin-3 production by novel bicistronic interleukin-3 reporter mice

doi: 10.1016/j.imlet.2020.02.006

Figure Lengend Snippet: Generation of IL-3-ZsGreen1 knock-in reporter (3Gr) mice using the CRISPR/Cas9 system. 3Gr mice were generated by inserting an internal ribosome entry site (IRES)-ZsGreen1 bicistronic cassette via CRISPR/Cas-stimulated homologous recombination directly into the 3′ untranslated region of the IL-3 gene. (A) Schematic of IL-3-IRES-ZsGreen1 donor vector. 5′ and 3′ homology regions (HR) are shown. (B) Schematic of wild-type IL-3 locus and IRES-ZsGreen1 knock-in allele. Positions of genotyping PCR primers (Il3-ScF1, Il3-ScR1, and IRES-R1) are indicated. (C) A representative gel image of PCR-based genotyping of a wild-type (WT) mouse and mice homozygous (3Gr) and heterozygous (Het) for the IRES-ZsGreen1 knock-in allele.

Article Snippet: IL-3 was detected with a PE anti-IL-3 mAb (clone MP2–8F8; 2 μg/ml; BD Biosciences, San Jose, CA).

Techniques: Knock-In, CRISPR, Generated, Homologous Recombination, Plasmid Preparation

Journal: Immunology letters

Article Title: Assessment of faithful interleukin-3 production by novel bicistronic interleukin-3 reporter mice

doi: 10.1016/j.imlet.2020.02.006

Figure Lengend Snippet: IL-3 secretion by 3Gr CD4 T cell subsets in vitro. Splenocytes from WT (n = 3) and 3Gr mice (n = 4) were pooled according to genotype and CD4 T cells purified and cultured under Th0, Th1, Th2, or Th17 conditions. After 5 days, cells were activated with anti-CD3/CD28 mAbs and supernatants collected after 24 h and analyzed for IL-3 by ELISA. Data presented are the mean ± SD of duplicate cultures. P = 0.068 for WT Th1 cells vs values for 3Gr Th1 cells. Data are representative of three independent experiments.

Article Snippet: IL-3 was detected with a PE anti-IL-3 mAb (clone MP2–8F8; 2 μg/ml; BD Biosciences, San Jose, CA).

Techniques: In Vitro, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Immunology letters

Article Title: Assessment of faithful interleukin-3 production by novel bicistronic interleukin-3 reporter mice

doi: 10.1016/j.imlet.2020.02.006

Figure Lengend Snippet: ZsGreen1 and intracellular IL-3 expression by 3Gr Th1 cells in vitro. Splenocytes from WT (n = 3) and 3Gr mice (n = 3) were pooled according to genotype and CD4 T cells purified and cultured under Th1 conditions. After 5 days, Th1 cells were activated 6 h with anti-CD3/CD28 mAbs and analyzed for ZsGreen1 and intracellular IL-3 expression by flow cytometry. Data are representative of three independent experiments.

Article Snippet: IL-3 was detected with a PE anti-IL-3 mAb (clone MP2–8F8; 2 μg/ml; BD Biosciences, San Jose, CA).

Techniques: Expressing, In Vitro, Purification, Cell Culture, Flow Cytometry

Journal: Cell metabolism

Article Title: Cholesterol Accumulation in Dendritic Cells Links the Inflammasome to Acquired Immunity

doi: 10.1016/j.cmet.2017.04.005

Figure Lengend Snippet: Control and DC-ABCDKO mice were 20 weeks old. Spleens were isolated, stained with the indicated antibodies, and analyzed by flow cytometry. (A–B) Gating strategy of splenic CD11c+MHCII+CD8a+ and CD11c+MHCII+CD11b+ DCs. (C) Total splenic CD8a+ and CD11b+ DCs. n=10. (D–E) Expression of MHCII on CD8a+ and CD11b+ DCs. n=6. (F–G) Antigen presentation. Mice were immunized with ova-peptide. At 24 h after injection, CD4+ OT-II cells from CD90.1 mice were isolated and labeled with CFSE. 2*106 cells were injected per mouse and 72 h after injection, CFSE dilution in CD90.1+CD4+ T-cells was assessed in spleen and inguinal lymph nodes. Representative CFSE dilutions are shown (F) and quantified (G). n=4. Data in C, E, and G are presented as mean ± SEM. **P<0.01, by t-test. See also Figure S4.

Article Snippet: For analysis of DCs, splenic homogenates were stained with a cocktail of antibodies against F480-PE-Cy7, MHCII-FITC (eBioscience), CD11c-PerCP-Cy5.5 (BD Pharmingen), CD11b-APC, CD8-APC-Cy7, CD86-PE, PD-L1-PE (eBioscience) and CD80-PB (Biolegend), or the common β subunit for the IL-3/GM-CSF receptor (BD Pharmingen).

Techniques: Isolation, Staining, Flow Cytometry, Expressing, Injection, Labeling

Journal: Cell metabolism

Article Title: Cholesterol Accumulation in Dendritic Cells Links the Inflammasome to Acquired Immunity

doi: 10.1016/j.cmet.2017.04.005

Figure Lengend Snippet: Mice used in all experiments were 20 weeks old unless indicated otherwise. (A–D). DC-Abca1/g1 deficiency enhances pro-IL-1β protein (A–B) and mRNA levels (C) and Nlrp3 mRNA levels (D) in splenic CD11c+MHCII+CD11b+ cells, indicative of inflammasome priming. FACS plot (A) and quantification (B) are shown. n=4. From mice of the indicated genotypes, CD11b+ cells were isolated from spleen (E–F) or LNs (G–H) using CD11b positive beads. Cells were lysed, and caspase-1 cleavage was assessed using Western blot. Representative images (E, G) are shown and caspase-1 cleavage was calculated as the ratio cleaved caspase-1 (p20) to pro-caspase-1 (p45) (F, H). n=4. (I) IL-18 plasma levels were assessed in 40 week old DC-ABCDKO and control mice using ELISA. Each datapoint indicates a single mouse. n=7–10. (J–K) CD11b+ cells were isolated from the spleens of control and DC-ABCDKO mice using CD11b positive beads, and incubated with or without rHDL (o/n). Media was collected and the secretion of IL-1β (J) and IL-18 (K) was assessed using ELISA, and corrected for cell protein. n=4. (L) Splenic cell homogenates were stained with an antibody to CD4, fixed and permeabilized, and stained for Tbet (L), or its isotype control. For quantification, values were corrected for their respective isotype controls. n=5. Data in B-D, F, and H, J–L are presented as mean ± SEM. B–D, F, I, and L, *P<0.05, **P<0.01, ***P<0.001, by t-test. H, J, K, *P<0.05, **P<0.01, ***P<0.001, by oneway ANOVA with Bonferroni post-test. See also Figure S5.

Article Snippet: For analysis of DCs, splenic homogenates were stained with a cocktail of antibodies against F480-PE-Cy7, MHCII-FITC (eBioscience), CD11c-PerCP-Cy5.5 (BD Pharmingen), CD11b-APC, CD8-APC-Cy7, CD86-PE, PD-L1-PE (eBioscience) and CD80-PB (Biolegend), or the common β subunit for the IL-3/GM-CSF receptor (BD Pharmingen).

Techniques: Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Staining