Journal: Scientific Reports

Article Title: Effect of IL-34 on T helper 17 cell proliferation and IL-17 secretion by peripheral blood mononuclear cells from rheumatoid arthritis patients

doi: 10.1038/s41598-020-79312-z

Figure Lengend Snippet: List of the sequence of gene primers.

Article Snippet: Intracellular cytokines were stained with BV421-labelled anti-IFN-γ (B27, BD Biosciences, USA), PE-labelled anti-IL-4 (8D4-8, BD Biosciences, USA) and AF647-labelled anti-IL-17A (N49-653, BD Biosciences, USA) antibodies for 30 min at 4 °C in the dark.

Techniques: Sequencing

Journal: Scientific Reports

Article Title: Effect of IL-34 on T helper 17 cell proliferation and IL-17 secretion by peripheral blood mononuclear cells from rheumatoid arthritis patients

doi: 10.1038/s41598-020-79312-z

Figure Lengend Snippet: The frequency of Th17 cells stimulated with different concentrations of IL-34 by flow cytometry quantification. ( A ) The gating strategy for Th17 cells in PBMCs from the RA patient. ( B ) Pseudo color plots show representative flow cytometric data of CD4 + IL-17 + T cells. Bar charts show the frequency of Th17 cell. The differences between four groups were tested by ANOVA and after post Tukey test. PBMCs were stimulated with phorbol myristate acetate and ionomycin for 4 h with Golgi Plug added for the last hour before flow cytometry assay.

Article Snippet: Intracellular cytokines were stained with BV421-labelled anti-IFN-γ (B27, BD Biosciences, USA), PE-labelled anti-IL-4 (8D4-8, BD Biosciences, USA) and AF647-labelled anti-IL-17A (N49-653, BD Biosciences, USA) antibodies for 30 min at 4 °C in the dark.

Techniques: Flow Cytometry

Journal: Scientific Reports

Article Title: Effect of IL-34 on T helper 17 cell proliferation and IL-17 secretion by peripheral blood mononuclear cells from rheumatoid arthritis patients

doi: 10.1038/s41598-020-79312-z

Figure Lengend Snippet: The differences in the gene expression levels of RORC ( A ), IL-17 ( B ), TBX21 ( C ), GATA3 ( D ), Foxp3 ( E ) and IL-10 ( F ) stimulated with different concentrations of IL-34 by reverse transcription-PCR analysis. PBMCs were stimulated with anti-CD3 and anti-CD28 for 3 days before RNA extraction. The differences between four groups were tested by ANOVA and after post Tukey test.

Article Snippet: Intracellular cytokines were stained with BV421-labelled anti-IFN-γ (B27, BD Biosciences, USA), PE-labelled anti-IL-4 (8D4-8, BD Biosciences, USA) and AF647-labelled anti-IL-17A (N49-653, BD Biosciences, USA) antibodies for 30 min at 4 °C in the dark.

Techniques: Expressing, RNA Extraction

Journal: Scientific Reports

Article Title: Effect of IL-34 on T helper 17 cell proliferation and IL-17 secretion by peripheral blood mononuclear cells from rheumatoid arthritis patients

doi: 10.1038/s41598-020-79312-z

Figure Lengend Snippet: The differences between the IL-17 ( A ), IFN-γ ( B ) and IL-10 ( C ) expression stimulated with different concentrations of IL-34 by ELISA. Cell culture supernatants of PBMCs treated with different concentrations of IL-34 and anti-CD3 and anti-CD28 for 3 days were used. The differences between four groups were tested by ANOVA and after post Tukey test.

Article Snippet: Intracellular cytokines were stained with BV421-labelled anti-IFN-γ (B27, BD Biosciences, USA), PE-labelled anti-IL-4 (8D4-8, BD Biosciences, USA) and AF647-labelled anti-IL-17A (N49-653, BD Biosciences, USA) antibodies for 30 min at 4 °C in the dark.

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

Journal: Mediators of Inflammation

Article Title: Disruption of Tumor Necrosis Factor Receptor-Associated Factor 5 Exacerbates Murine Experimental Colitis via Regulating T Helper Cell-Mediated Inflammation

doi: 10.1155/2016/9453745

Figure Lengend Snippet: The gene-specific primer pairs list in the study.

Article Snippet: The following primary antibodies were used: anti-IFN- γ (1 : 200 dilution; Proteintech, USA), anti-IL-4 (1 : 100 dilution; BioLegend, USA), anti-IL-17a (1 : 200 dilution; Proteintech, USA), anti-p65 (1 : 100 dilution; Cell Signaling Technology, USA), anti-CD4 (1 : 100 dilution; BioLegend, USA), and anti-CK18 (1 : 100 dilution; Goodbio Technology Co., China).

Techniques:

Journal: Mediators of Inflammation

Article Title: Disruption of Tumor Necrosis Factor Receptor-Associated Factor 5 Exacerbates Murine Experimental Colitis via Regulating T Helper Cell-Mediated Inflammation

doi: 10.1155/2016/9453745

Figure Lengend Snippet: Analysis of the mRNA expression of cytokines and transcription factors for Th cells in the colons of TRAF5 KO and WT mice. (a) TNF- α , (b) IFN- γ , (c) IL-4, (d) IL-17a, (e) IL-22, (f) T-bet, (g) GATA-3, (h) ROR- α , and (i) ROR- γ t mRNA expression levels were determined by real-time PCR. The data are representative of 3 independent experiments (mean ± SD). N = 8 per group. ∗ p < 0.05 versus the DSS-treated WT group; ∗∗ p < 0.01 versus the DSS-treated WT group. NS: not significant versus the DSS-treated WT group.

Article Snippet: The following primary antibodies were used: anti-IFN- γ (1 : 200 dilution; Proteintech, USA), anti-IL-4 (1 : 100 dilution; BioLegend, USA), anti-IL-17a (1 : 200 dilution; Proteintech, USA), anti-p65 (1 : 100 dilution; Cell Signaling Technology, USA), anti-CD4 (1 : 100 dilution; BioLegend, USA), and anti-CK18 (1 : 100 dilution; Goodbio Technology Co., China).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Mediators of Inflammation

Article Title: Disruption of Tumor Necrosis Factor Receptor-Associated Factor 5 Exacerbates Murine Experimental Colitis via Regulating T Helper Cell-Mediated Inflammation

doi: 10.1155/2016/9453745

Figure Lengend Snippet: Deletion of TRAF5 increases the protein expressions of the cytokines IFN- γ , IL-4, and IL-17a in the colons of mice induced by DSS. (a) The production of IFN- γ , IL-4, and IL-17a in the colons of DSS-induced mice or control mice was examined by ELISA ( N = 6 per group). (b) Representative immunofluorescence staining for IFN- γ , IL-4, and IL-17a in the colons of TRAF5 KO and WT mice (magnification 400x). (c) Quantification of positive cells ( N = 4 per group). The data are representative of 3 independent experiments (mean ± SD). ∗ p < 0.05 versus the DSS-treated WT group; ∗∗ p < 0.01 versus the DSS-treated WT group.

Article Snippet: The following primary antibodies were used: anti-IFN- γ (1 : 200 dilution; Proteintech, USA), anti-IL-4 (1 : 100 dilution; BioLegend, USA), anti-IL-17a (1 : 200 dilution; Proteintech, USA), anti-p65 (1 : 100 dilution; Cell Signaling Technology, USA), anti-CD4 (1 : 100 dilution; BioLegend, USA), and anti-CK18 (1 : 100 dilution; Goodbio Technology Co., China).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Mediators of Inflammation

Article Title: Disruption of Tumor Necrosis Factor Receptor-Associated Factor 5 Exacerbates Murine Experimental Colitis via Regulating T Helper Cell-Mediated Inflammation

doi: 10.1155/2016/9453745

Figure Lengend Snippet: Increased proportions of Th2 and IFN- γ /IL-17a-coproducing CD4 + T cells in the colons of TRAF5-deficient animals induced by DSS. LPMCs obtained from the colons of DSS-fed KO and WT mice were stimulated with PMA/ionomycin and subjected to surface staining for CD4. After fixation and permeabilization, intracellular staining for IL-4, IFN- γ , and IL-17a was performed as described in . CD4 + T cells were then gated and analyzed. (a) Representative flow plots. The percentages of (b) Th2 cells (CD4 + IL-4 + ), (c) IFN- γ /IL-17a-coproducing CD4 + T cells (CD4 + IFN- γ + IL-17a + ), (d) classical Th1 cells (CD4 + IFN- γ + IL-17a − ), and (e) classical Th17 cells (CD4 + IL-17a + IFN- γ − ) are presented as the mean ± SD. The data are representative of 3 independent experiments. N = 6 per group. ∗∗ p < 0.01 versus the DSS-treated WT group. NS: not significant versus the DSS-treated WT group.

Article Snippet: The following primary antibodies were used: anti-IFN- γ (1 : 200 dilution; Proteintech, USA), anti-IL-4 (1 : 100 dilution; BioLegend, USA), anti-IL-17a (1 : 200 dilution; Proteintech, USA), anti-p65 (1 : 100 dilution; Cell Signaling Technology, USA), anti-CD4 (1 : 100 dilution; BioLegend, USA), and anti-CK18 (1 : 100 dilution; Goodbio Technology Co., China).

Techniques: Staining

Journal: Journal of Neuroinflammation

Article Title: RhANP attenuates endotoxin-derived cognitive dysfunction through subdiaphragmatic vagus nerve-mediated gut microbiota–brain axis

doi: 10.1186/s12974-021-02356-z

Figure Lengend Snippet: Roles of SDV in rhANP-mediated reduction of LPS-induced systemic inflammation and neuroinflammation. a Treatment schedule. Mice were intraperitoneally injected with lipopolysaccharides (LPS, 5 mg/kg). Recombinant human ANP (rhANP; 1.0 mg/kg) or 0.9% saline were intraperitoneally injected to mice at 24 h before and 10 min after LPS injection. Subdiaphragmatic vagotomy (SDV) was performed 14 days prior to LPS injection. Plasma and hippocampus were collected 24 h after LPS injection. b Body weight loss in each group (two-way ANOVA: rhANP: F 1,36 = 7.058, P = 0.0117; SDV: F 1,36 = 12.72, P = 0.0010; interaction: F 1,36 = 4.923, P = 0.0329). The plasma levels of interleukin (IL)-6 ( c ; two-way ANOVA: rhANP: F 1,36 = 13.07, P = 0.0009; SDV: F 1,36 = 15.60, P = 0.0003; interaction: F 1,36 = 20.51, P < 0.0001), IL-17A ( d ; two-way ANOVA: rhANP: F 1,36 = 6.111, P = 0.0183; SDV: F 1,36 = 7.794, P = 0.0083; interaction: F 1,36 = 6.027, P = 0.0191), interferon (IFN)-γ ( e ; two-way ANOVA: rhANP: F 1,36 = 6.460, P = 0.0155; SDV: F 1,36 = 8.447, P = 0.0062; interaction: F 1,36 = 7.836, P = 0.0082), and tumor necrosis factor (TNF)-α ( f ; two-way ANOVA: rhANP: F 1,36 = 4.828, P = 0.0345; SDV: F 1,36 = 6.217, P = 0.0174; interaction: F 1,36 = 7.883, P = 0.0080). Western blot analysis of ionized calcium-binding adapter molecule 1 (iba-1) ( g ; two-way ANOVA: rhANP: F 1,36 = 6.208, P = 0.0175; SDV: F 1,36 = 4.772, P = 0.0355; interaction: F 1,36 = 4.996, P = 0.0317), IL-6 ( h ; two-way ANOVA: rhANP: F 1,36 = 6.286, P = 0.0168; SDV: F 1,36 = 6.894, P = 0.0126; interaction: F 1,36 = 8.569, P = 0.0059), IL-17A ( i ; two-way ANOVA: rhANP: F 1,36 = 4.268, P = 0.0461; SDV: F 1,36 = 3.363, P = 0.0750; interaction: F 1,36 = 5.503, P = 0.0246), interferon (IFN)-γ ( j ; two-way ANOVA: rhANP: F 1,36 = 4.706, P = 0.0367; SDV: F 1,36 = 5.685, P = 0.0225; interaction: F 1,36 = 3.530, P = 0.0684), TNF-α ( k ; two-way ANOVA: rhANP: F 1,36 = 1.598, P = 0.2144; SDV: F 1,36 = 12.72, P = 0.0010; interaction: F 1,36 = 4.345, P = 0.0443), inducible nitric oxide synthase (iNOS) ( l ; two-way ANOVA: rhANP: F 1,36 = 4.764, P = 0.0357; SDV: F 1,36 = 2.933, P = 0.0954; interaction: F 1,36 = 5.462, P = 0.0251) and their respective β-actin in the hippocampus. Data are shown as mean ± SEM, n = 10/group. * P < 0.05, ** P < 0.01, *** P < 0.0001; N.S. not significant

Article Snippet: The membranes were blocked in 5% non-fat dried milk for 1 h at room temperature, and then incubated with the following primary antibodies: rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (iba-1; 1:1000, #016-20001: Wako Pure Chemical Industries, Ltd., Tokyo, Japan), rabbit monoclonal anti-IL-6 (1:1000, #12912S, Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-IL-17A (1:1000, #A0688, ABclonal, Wuhan, China), rabbit monoclonal anti-IFN-γ (1:1000, #A12450, ABclonal, Wuhan, China), rabbit polyclonal anti-TNF-α (1:1000, #A0277, ABclonal, Wuhan, China), rabbit monoclonal anti-inducible nitric oxide synthase (iNOS; 1:1000, #ab3523, Abcam, Cambridge, MA, USA), brain-derived neurotrophic factor (BDNF; 1:1000, #A4873, ABclonal, Wuhan, China), rabbit polyclonal phosphorylated tyrosine kinase receptor B (p-TrkB; 1:1000, #AP0423, ABclonal, Wuhan, China), rabbit polyclonal total TrkB (t-TrkB; 1:1000, #A2099, ABclonal, Wuhan, China), and mouse monoclonal anti-β-actin (1:1000, #A2319, ABclonal, Wuhan, China) overnight at 4 ℃.

Techniques: Injection, Recombinant, Western Blot, Binding Assay

Journal: Journal of Neuroinflammation

Article Title: RhANP attenuates endotoxin-derived cognitive dysfunction through subdiaphragmatic vagus nerve-mediated gut microbiota–brain axis

doi: 10.1186/s12974-021-02356-z

Figure Lengend Snippet: Effects of rhANP on plasma inflammatory cytokines after LPS-triggered endotoxemia. a Treatment schedule. Mice were intraperitoneally injected with lipopolysaccharides (LPS, 5 mg/kg) or 0.9% saline. Recombinant human ANP (rhANP; 1.0 mg/kg) or 0.9% saline were intraperitoneally injected to mice 24 h before and 10 min after LPS injection. Spleen and plasma were collected 24 h after injection of LPS or 0.9% saline. b Body weight loss in mice treated with rhANP or 0.9% saline 24 h after injection of LPS or 0.9% saline (one-way ANOVA: F 2,27 = 58.21, P < 0.0001). c Representative picture of spleen and spleen weight (one-way ANOVA: F 2,28 = 27.53, P < 0.0001). d Ratio of spleen weight/body weight (one-way ANOVA: F 2,28 = 20.17, P < 0.0001). e Plasma levels of interleukin (IL)-6 (one-way ANOVA: F 2,28 = 65.35, P < 0.0001). f Plasma levels of IL-17A (one-way ANOVA: F 2,28 = 16.82, P < 0.0001). g Plasma levels of interferon (IFN)-γ (one-way ANOVA: F 2,28 = 12.62, P < 0.0001). h Plasma levels of tumor necrosis factor (TNF)-α (one-way ANOVA: F 2,28 = 51.43, P < 0.0001). i There was a positive correlation (r = 0.797, P < 0.001) between spleen weight and plasma IL-6. j There was a positive correlation ( r = 0.629, P < 0.001) between spleen weight and plasma IL-17A. k There was a positive correlation ( r = 0.479, P = 0.006) between spleen weight and plasma IFN-γ. l Positive correlation ( r = 0.637, P < 0.001) between spleen weight and plasma TNF-α was observed. Data are shown as mean ± SEM, n = 10 or 11/group. ** P < 0.01, *** P < 0.0001

Article Snippet: The membranes were blocked in 5% non-fat dried milk for 1 h at room temperature, and then incubated with the following primary antibodies: rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (iba-1; 1:1000, #016-20001: Wako Pure Chemical Industries, Ltd., Tokyo, Japan), rabbit monoclonal anti-IL-6 (1:1000, #12912S, Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-IL-17A (1:1000, #A0688, ABclonal, Wuhan, China), rabbit monoclonal anti-IFN-γ (1:1000, #A12450, ABclonal, Wuhan, China), rabbit polyclonal anti-TNF-α (1:1000, #A0277, ABclonal, Wuhan, China), rabbit monoclonal anti-inducible nitric oxide synthase (iNOS; 1:1000, #ab3523, Abcam, Cambridge, MA, USA), brain-derived neurotrophic factor (BDNF; 1:1000, #A4873, ABclonal, Wuhan, China), rabbit polyclonal phosphorylated tyrosine kinase receptor B (p-TrkB; 1:1000, #AP0423, ABclonal, Wuhan, China), rabbit polyclonal total TrkB (t-TrkB; 1:1000, #A2099, ABclonal, Wuhan, China), and mouse monoclonal anti-β-actin (1:1000, #A2319, ABclonal, Wuhan, China) overnight at 4 ℃.

Techniques: Injection, Recombinant

Journal: Journal of Neuroinflammation

Article Title: RhANP attenuates endotoxin-derived cognitive dysfunction through subdiaphragmatic vagus nerve-mediated gut microbiota–brain axis

doi: 10.1186/s12974-021-02356-z

Figure Lengend Snippet: Effects of rhANP on the neuroinflammation and cognitive function after LPS-triggered endotoxemia. a Treatment schedule. Mice were intraperitoneally injected with lipopolysaccharides (LPS, 5 mg/kg) or 0.9% saline. Recombinant human ANP (rhANP; 1.0 mg/kg) or 0.9% saline were intraperitoneally injected to mice 24 h before and 10 min after LPS injection. The prefrontal cortex (PFC) and hippocampus were collected 24 h after injection of LPS or 0.9% saline. b , c Western blot analysis of ionized calcium-binding adapter molecule 1 (iba-1) in the prefrontal cortex (PFC) (one-way ANOVA: F 2,27 = 6.170, P = 0.0062) and hippocampus (one-way ANOVA: F 2,27 = 5.250, P = 0.0119). d , e Western blot analysis of interleukin (IL)-6 in the PFC (one-way ANOVA: F 2,27 = 5.958, P = 0.0072) and hippocampus (one-way ANOVA: F 2,27 = 17.60, P < 0.0001). f , g Western blot analysis of IL-17A in the PFC (one-way ANOVA: F 2,27 = 4.498, P = 0.0206) and hippocampus (one-way ANOVA: F 2,27 = 6.268, P = 0.0058). h , i Western blot analysis of interferon (IFN)-γ in the PFC (one-way ANOVA: F 2,27 = 11.18, P = 0.0003) and hippocampus (one-way ANOVA: F 2,27 = 7.611, P = 0.0024). j , k Western blot analysis of tumor necrosis factor (TNF)-α in the PFC (one-way ANOVA: F 2,27 = 5.530, P = 0.0097) and hippocampus (one-way ANOVA: F 2,27 = 8.550, P = 0.0013). l , m Western blot analysis of inducible nitric oxide synthase (iNOS) in the PFC (one-way ANOVA: F 2,27 = 7.328, P = 0.0029) and hippocampus (one-way ANOVA: F 2,27 = 7.597, P = 0.0024). n , o Entries in the novel arm (one-way ANOVA: F 2,27 = 31.33, P < 0.0001) and duration in the novel arm (one-way ANOVA: F 2,27 = 13.34, P < 0.0001) in the Y maze test. p Latency to eat food in the buried food test (one-way ANOVA: F 2,27 = 7.129, P = 0.0033). Data are shown as mean ± SEM, n = 10/group. * P < 0.05, ** P < 0.01, *** P < 0.0001; N.S. not significant

Article Snippet: The membranes were blocked in 5% non-fat dried milk for 1 h at room temperature, and then incubated with the following primary antibodies: rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (iba-1; 1:1000, #016-20001: Wako Pure Chemical Industries, Ltd., Tokyo, Japan), rabbit monoclonal anti-IL-6 (1:1000, #12912S, Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-IL-17A (1:1000, #A0688, ABclonal, Wuhan, China), rabbit monoclonal anti-IFN-γ (1:1000, #A12450, ABclonal, Wuhan, China), rabbit polyclonal anti-TNF-α (1:1000, #A0277, ABclonal, Wuhan, China), rabbit monoclonal anti-inducible nitric oxide synthase (iNOS; 1:1000, #ab3523, Abcam, Cambridge, MA, USA), brain-derived neurotrophic factor (BDNF; 1:1000, #A4873, ABclonal, Wuhan, China), rabbit polyclonal phosphorylated tyrosine kinase receptor B (p-TrkB; 1:1000, #AP0423, ABclonal, Wuhan, China), rabbit polyclonal total TrkB (t-TrkB; 1:1000, #A2099, ABclonal, Wuhan, China), and mouse monoclonal anti-β-actin (1:1000, #A2319, ABclonal, Wuhan, China) overnight at 4 ℃.

Techniques: Injection, Recombinant, Western Blot, Binding Assay

Journal: Mucosal Immunology

Article Title: IL-35 is critical in suppressing superantigenic Staphylococcus aureus -driven inflammatory Th17 responses in human nasopharynx-associated lymphoid tissue

doi: 10.1038/s41385-019-0246-1

Figure Lengend Snippet: a , b , d , e Intracellular cytokine analysis of IL-17A-expressing CD4 + T cells (Th17) in isolated human tonsillar MNCs 48 h following bacterial CCS (1 µg/ml) stimulation, compared to media control (MC) MNCs. a Dot plots were gated on CD4 + T cells and numbers in the top right quadrants indicate the percentage of Th17 cells within the CD4 + T cell population. Data were analyzed using paired t -test and displayed in mean ± SEM, n = 10. b Dose-dependent Th17 responses activated by NonSAg- Sau and SAg- Sau, respectively. Results are representative of 3 individual samples. c IL-17A concentration in tonsillar MNCs culture supernatants were measured by ELISA and samples assayed in duplicates. Data displayed is individual data points with mean ± SEM, n = 10. Paired t -test was performed on log-transformed data. d Box and Whiskers plot showing the percentage of Th17 cells within CD4 + T cells in tonsillar MNCs stimulated with Spn, M. catarrhalis , coagulase-negative staphylococcus (CNS, C4 and C5) and SAg- Sau, respectively. e The percentage of Th17 cells within CD4 + T cell population was summarized for tonsillar MNCs activated by NonSAg- Sau , SAg- Sau, and Sau carriage strains (C1, C2, and C3). Data ( d, e ) was displayed in median (center line), upper and lower quartiles (box limits) and minimum to maximum range (whiskers). 8 ( d ) and 5 ( e ) individual samples were tested and analyzed. f Staphylococcal enterotoxin A-E level in Sau strains (PC, positive control. NC, negative control), test was performed in duplicate. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Anti-human CD4-PECy7, IL-17A-Alexa Fluor 647, Foxp3-Alexa Fluor 488, IL-10-PE, and RORγt-PE FACS antibodies were from BD Biosciences.

Techniques: Expressing, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transformation Assay, Positive Control, Negative Control

Journal: Mucosal Immunology

Article Title: IL-35 is critical in suppressing superantigenic Staphylococcus aureus -driven inflammatory Th17 responses in human nasopharynx-associated lymphoid tissue

doi: 10.1038/s41385-019-0246-1

Figure Lengend Snippet: a Analysis of Treg expansion and IL-17A-expressing Tregs in isolated human tonsillar MNCs 48 h following bacterial CCS (1 µg/ml) stimulation. Dot plots were gated on CD4 + T cells and numbers in bottom right and top right quadrants indicate the percentage of IL-17A − Foxp3 + and IL-17A + Foxp3 + cells, respectively, within the CD4 + T cell population. Data is displayed as mean ± SEM by column bar graphs and analyzed using paired t -test, n = 10. b Unfractionated MNCs and CD25 + cell depleted MNCs were labeled with CFSE in order to quantify CD4 + T cell proliferation 5 days after SAg- Sau CCS (1 µg/ml) stimulation. Histogram plots were gated on CD4 + T cells showing the median fluorescent intensity (MFI) of CFSE and numbers in the top left corners indicate percentage of proliferated CD4 + T cells. Data points with mean ± SEM are shown in the dot plot on the right with 3 individual samples tested. c, d IL-17A and IFNγ expression after 48 h of SAg- Sau CCS (1 µg/ml) stimulation for unfractionated MNCs and CD25 + cell depleted MNCs. Results were summarized from 3 individual samples. c Zebra plots were gated on lymphocytes and numbers in top left and right quadrants indicate the percentage of IFNγ + CD4 − lymphocytes and IFNγ + CD4 + T cells (Th1) in total lymphocytes, respectively. d Zebra plots were gated on CD4 + T cells, with the percentage of Th17 cells in CD4 + T cell population shown in the top right quadrants. Data were analyzed using paired t -test. (ns: not significant).

Article Snippet: Anti-human CD4-PECy7, IL-17A-Alexa Fluor 647, Foxp3-Alexa Fluor 488, IL-10-PE, and RORγt-PE FACS antibodies were from BD Biosciences.

Techniques: Expressing, Isolation, Labeling

Journal: Mucosal Immunology

Article Title: IL-35 is critical in suppressing superantigenic Staphylococcus aureus -driven inflammatory Th17 responses in human nasopharynx-associated lymphoid tissue

doi: 10.1038/s41385-019-0246-1

Figure Lengend Snippet: a, b Analysis of IL-17A or IFNγ expressing CD4 + T cell responses in SAg- Sau stimulated tonsillar MNCs treated with IL-10 neutralizing antibody or isotype control. MC is unstimulated media control. Zebra plots were gated on CD4 + T cells and numbers in top right quadrants indicate the percentages of Th1 ( a ) or Th17 ( b ) within CD4 + T cell population. Data was analyzed using paired t -test with p values indicated, n = 7. c , d CD69 + cell depleted tonsillar MNCs were stimulated with SAg- Sau CCS in the presence or absence of recombinant IL-10 (10 ng/ml) for 48 h. Ctrl is the stimulation control. c Numbers in top right quadrants of the zebra plots indicate the percentage of Th17 in CD4 + T cells. Results of 8 individual samples were analyzed and summarized in symbol and line plot. d The percentage of IL-17A + IFNγ - , IL-17A + IFNγ + , and IL-17A - IFNγ + cells within CD4 + T cell population is shown in top left, top right and bottom right quadrants of the zebra plots. Summarized data is displayed in symbol and line plot, n = 5.

Article Snippet: Anti-human CD4-PECy7, IL-17A-Alexa Fluor 647, Foxp3-Alexa Fluor 488, IL-10-PE, and RORγt-PE FACS antibodies were from BD Biosciences.

Techniques: Expressing, Recombinant

Journal: Mucosal Immunology

Article Title: IL-35 is critical in suppressing superantigenic Staphylococcus aureus -driven inflammatory Th17 responses in human nasopharynx-associated lymphoid tissue

doi: 10.1038/s41385-019-0246-1

Figure Lengend Snippet: a–c Tonsillar MNCs were stimulated with SAg- Sau CCS for 48 h with 100 ng/ml of recombinant IL-35 or Fc control protein. a The association between fold increase in Th17 proportion and Th17 inhibition by IL-35. Each dot represents an individual sample, n = 22. Data was analyzed using linear regression. b The proportion of Th17 cells in total CD4 + T cells is shown as fold increase against media control (MC). The line plot shows the suppressive effect of IL-35 on Th17 responses in high responder group (≥5-fold) and low responder group (<5-fold), respectively, n = 8 per group. c IL-17A concentration in MNC culture supernatant of the high responders ( n = 8) measured by ELISA, tests were performed in duplicate. d Inhibition of SAg- Sau activated Th17 and Th1 responses by IL-35 or IL-10 in tonsillar MNCs. Zebra plots were gated on CD4 + T cells and numbers in top left and bottom right quadrants indicate the percentage of IL-17A + IFNγ − and IL-17A − IFNγ + cells respectively in CD4 + T cell population. Data of 5 individual samples was analyzed using paired t -test and summarized in bar chats. e , f Tonsillar MNCs were stimulated with SAg- Sau (1 µg/ml) in the presence of conditioned medium from IL-35-transfected CHO cells (Clone 7) or control CHO cells at 1, 2, and 10% respectively. Five individual samples were tested and analyzed. e Representative dot plots were gated on CD4 + T cells and numbers in top right quadrants indicate the percentage of IL-17A + Ki67 + cells (Proliferating Th17 cells) in CD4 + T cells. f Suppression (%) of Th17 proliferation in IL-35-transfected CHO cell medium-treated or control CHO cell medium-treated MNCs was calculated against stimulated MNCs without CHO cell medium, mean ± SEM is shown.

Article Snippet: Anti-human CD4-PECy7, IL-17A-Alexa Fluor 647, Foxp3-Alexa Fluor 488, IL-10-PE, and RORγt-PE FACS antibodies were from BD Biosciences.

Techniques: Recombinant, Inhibition, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Mucosal Immunology

Article Title: IL-35 is critical in suppressing superantigenic Staphylococcus aureus -driven inflammatory Th17 responses in human nasopharynx-associated lymphoid tissue

doi: 10.1038/s41385-019-0246-1

Figure Lengend Snippet: CD45RO + cell depleted tonsillar MNCs were stimulated with SAg- Sau CCS (50 ng/ml) for 7 days in the presence of recombinant IL-1β (50 ng/ml), IL-21 (50 ng/ml), and TGFβ1 (2 ng/ml). 10 ng/ml of recombinant IL-35 or Fc control protein were added at day 0 and day 3. a IL-17A concentration in the cell culture supernatants as measured by ELISA, tests were performed in duplicate. Results of 6 individual samples were analyzed using paired t -test. b Line plot showing the percentage of RORγt + CD4 − lymphocytes and RORγt + CD4 + T cells in SAg- Sau stimulated tonsillar MNCs with or without IL-35. Data is shown in mean ± SEM, n = 4.

Article Snippet: Anti-human CD4-PECy7, IL-17A-Alexa Fluor 647, Foxp3-Alexa Fluor 488, IL-10-PE, and RORγt-PE FACS antibodies were from BD Biosciences.

Techniques: Recombinant, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay