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: The Journal of Allergy and Clinical Immunology

Article Title: Cysteinyl leukotriene E 4 activates human group 2 innate lymphoid cells and enhances the effect of prostaglandin D 2 and epithelial cytokines

doi: 10.1016/j.jaci.2016.12.958

Figure Lengend Snippet: ILC2 isolation. ILC2s isolated from human blood were lineage marker negative (CD3, CD14, CD19, CD56, CD11c, CD11b, FcεRI, TCRγδ, TCRα,β and CD123), CD45 high , IL-7 receptor α–positive, and CRTH2 + cells. FSC , Forward scatter.

Article Snippet: Anti-human CD123 antibody was from R&D Systems, and anti-human CysLTR1 antibody was from Novus Biologicals (Littleton, Colo).

Techniques: Isolation, Marker

Journal: Nature Communications

Article Title: Osteoprotegerin-dependent M cell self-regulation balances gut infection and immunity

doi: 10.1038/s41467-019-13883-y

Figure Lengend Snippet: a Enrichment analysis based on KEGG functional hierarchy for gene expression in M cells relative to their expression in enterocytes. Node size indicates the false-discovery rate of the parametric enrichment analysis. Red and blue nodes indicate respective significantly upregulated and downregulated pathways in M cells. b Gene expression profiles of enterocytes and M cells are shown. The heat map colors represent logFC for expression levels of genes compared with the mean expression value of each gene in enterocytes. c Increased expression of Tnfrsf11b ( Opg ) in the FAE of Peyer’s patches, relative to expression in villus epithelium (VE). Results are normalized to Gapdh expression and are presented relative to the expression in the mean of VE. Values are presented as the mean ± standard error. *** p < 0.005 (Student’s t -test, n = 4 animals). d Stereomicroscopic image of a whole-mount specimen of FAE monolayer and associated crypts, which was separated from the follicle by manipulation after treatment with EDTA as described in the Methods. The FAE area was surrounded by 12 crypts, as indicated by arrows. Scale bar: 200 µm. e OPG (red) is expressed in Tnfaip2 (blue)-positive and GP2 (green)-positive M cells in the FAE. Scale bars: 50 µm. f OPG (green) is expressed in Spi-B (red)-positive M cells of a FAE-associated crypt. The lower panel is an enlarged view of the area within the square in the upper panel. Cell nuclei were stained with DAPI (blue, left panel). F-actin was stained with phalloidin (gray, right panel). DIC differential interference contrast image. Scale bars: upper panel, 20 µm; lower panel, 10 µm. g Opg is an early expressing gene in the ileal epithelium after RANKL administration. Results were normalized to Gapdh expression and are presented relative to the expression in the epithelium without GST-RANKL treatment (time 0). Values are presented as the mean ± standard error. All data are representative of two ( c ) or three independent experiments ( a , b , d , e – g ). The source data underlying panels c and g are provided as a Source Data file.

Article Snippet: The primary antibodies used for immunofluorescence staining are as follows: anti-OPG antibody (catalog number AF549; R&D systems, 1:400); anti-GP2 antibody (D278-3; MBL, 1:400); anti-Tnfaip2 antibody (Kimura et al. : 200); anti-SpiB antibody (AF7204; R&D Systems, 1:200); anti-RelB antibody (sc-226; Santa Cruz Biotechnology, 1:100); and anti-RANKL antibody (14-5952; eBioscience, 1:100).

Techniques: Functional Assay, Gene Expression, Expressing, Staining

Journal: Nature Communications

Article Title: Osteoprotegerin-dependent M cell self-regulation balances gut infection and immunity

doi: 10.1038/s41467-019-13883-y

Figure Lengend Snippet: a The numbers of M cells labeled with Tnfaip2 (green) and GP2 (red) are prominently increased in the follicle-associated epithelium (FAE) of Payer’s patches from Opg −/− mice. Each indicated channel is shown separately in gray scale. Scale bars: 200 µm. b The numbers of Tnfaip2-positive or GP2-positive M cells in the FAE of Peyer’s patches from WT and Opg −/− mice were quantified. Values are presented as the mean ± standard deviation. *** p < 0.005 (Student’s t -test, n = 4 of WT and 9 of Opg −/− mice). c The expression levels of M cell-associated genes in the FAE of Peyer’s patches were normalized to Gapdh expression and are presented relative to the expression in the villus epithelium (VE). Values are presented as the mean ± standard deviation. ** p < 0.01, *** p < 0.005 (Student’s t -test, n = 4 animals). d Whole-mount staining images of GP2 (red), Spi-B (green), and DAPI (blue) in cecal FAE. Scale bars: 100 µm. e The numbers of Tnfaip2-positive or GP2-positive M cells in the FAE of cecal patches from WT and Opg −/− mice were quantified. Values are presented as the mean ± standard deviation. * p < 0.05 (Student’s t-test, n = 4 of WT and 9 of Opg −/− mice). f The uptake of latex beads into Peyer’s patches and cecal patches was measured. Data from three independent experiments are presented as the mean ± standard deviation. *** p < 0.005. n.s. not significant (Student’s t -test, n = 7 animals). The source data underlying panels b , c , e and f are provided as a Source Data file.

Article Snippet: The primary antibodies used for immunofluorescence staining are as follows: anti-OPG antibody (catalog number AF549; R&D systems, 1:400); anti-GP2 antibody (D278-3; MBL, 1:400); anti-Tnfaip2 antibody (Kimura et al. : 200); anti-SpiB antibody (AF7204; R&D Systems, 1:200); anti-RelB antibody (sc-226; Santa Cruz Biotechnology, 1:100); and anti-RANKL antibody (14-5952; eBioscience, 1:100).

Techniques: Labeling, Standard Deviation, Expressing, Staining

Journal: Nature Communications

Article Title: Osteoprotegerin-dependent M cell self-regulation balances gut infection and immunity

doi: 10.1038/s41467-019-13883-y

Figure Lengend Snippet: a Whole-mount immunostaining of the FAE of Peyer’s patches (left) and cecal patches (right) for OPG (green) and Spi-B (red). Nuclei were stained with DAPI (blue); scale bars: 50 µm. b Scatter plots of the fluorescence intensities of OPG versus Spi-B. Red dots represent cells stained with anti-Spi-B antibody that was conjugated with HyLyte Fluor 555 and anti-OPG antibody that was conjugated with HyLyte Fluor 647. Blue dots represent background fluorescence intensity of randomly selected non-stained cells. Fluorescence intensities were measured for at least 3000 cells from five FAEs of three mice. c Frequencies of OPG high M cells in Peyer’s patches and cecal patches were quantified. * p < 0.05, *** p < 0.005. Student’s t -test, n = 5 FAE from three animals. The source data underlying panels b and c are provided as a Source Data file.

Article Snippet: The primary antibodies used for immunofluorescence staining are as follows: anti-OPG antibody (catalog number AF549; R&D systems, 1:400); anti-GP2 antibody (D278-3; MBL, 1:400); anti-Tnfaip2 antibody (Kimura et al. : 200); anti-SpiB antibody (AF7204; R&D Systems, 1:200); anti-RelB antibody (sc-226; Santa Cruz Biotechnology, 1:100); and anti-RANKL antibody (14-5952; eBioscience, 1:100).

Techniques: Immunostaining, Staining, Fluorescence

Journal: Nature Communications

Article Title: Osteoprotegerin-dependent M cell self-regulation balances gut infection and immunity

doi: 10.1038/s41467-019-13883-y

Figure Lengend Snippet: a GP2 + cells (red) are more effectively induced in the cecal epithelium (CE) and ileal villi (VE) of Opg −/− mice by RANKL administration. Whole-mount immunohistochemical images of Spi-B (green) and GP2 (red) in the VE and CE of WT (upper panels) and Opg −/− mice (lower panels) treated with either GST (control; left) or GST-RANKL (right). Scale bars: 100 µm. Representative images from three independent experiment are shown. b Quantitative PCR analysis of M-cell marker expression in conventional epithelia from the VE and CE of mice injected with GST (control) or GST-RANKL. Results were normalized to Gapdh expression and are presented relative to the expression in the ileal epithelium from GST-treated mice. Data shown are mean values from three independent experiments (error bars indicate standard deviation). *** p < 0.005, ** p < 0.01, * p < 0.05; p values were calculated with the Student’s t -test ( n = 3 biologically independent experiments). c , d Nuclear translocation activities of RelB and p52 following RANKL stimulation are enhanced in Opg −/− mice. c Western blot analysis of p100/p52 and RelB in the VE and CE of mice injected with GST or GST-RANKL. Rpt4, a subunit of the 26S proteasome, was used as an internal control for the cytoplasmic fraction. Lamin A/C (Lamin) was used as an internal control for the nuclear fraction. Data are representative of two independent experiments. d Right, single confocal planes of the cecal FAE from WT and Opg −/− mice. FAE monolayers were stained with anti-RelB (green) and anti-Spi-B (red) antibodies and with Hoechst 33342. Left, a bar graph summarizing the proportions of RelB-positive cells among total numbers of M cells (Spi-B-positive cells). *** p < 0.005; p values were calculated with the Student’s t -test ( n = 4 biologically independent experiments). Scale bars: 20 µm. The source data underlying panels b and d and non-cropped scan images of western blotting ( c ) are provided as a Source Data file.

Article Snippet: The primary antibodies used for immunofluorescence staining are as follows: anti-OPG antibody (catalog number AF549; R&D systems, 1:400); anti-GP2 antibody (D278-3; MBL, 1:400); anti-Tnfaip2 antibody (Kimura et al. : 200); anti-SpiB antibody (AF7204; R&D Systems, 1:200); anti-RelB antibody (sc-226; Santa Cruz Biotechnology, 1:100); and anti-RANKL antibody (14-5952; eBioscience, 1:100).

Techniques: Immunohistochemical staining, Control, Real-time Polymerase Chain Reaction, Marker, Expressing, Injection, Standard Deviation, Translocation Assay, Western Blot, Staining