Journal: Cell reports

Article Title: TLR9 Sensing of Self-DNA Controls Cell-Mediated Immunity to Listeria Infection via Rapid Conversion of Conventional CD4 + T Cells to T reg

doi: 10.1016/j.celrep.2020.01.040

Figure Lengend Snippet: (A and B) Tlr2 , Tlr4 , Tlr7 , and Tlr9 mRNA normalized to Hprt1 mRNA in (A) whole spleen and (B) sorted DC and T cell populations in C57BL/6 mice infected with LD or HD Lm Δ actA -Ova (n = 3 per group). (C and D) p65-GFP knockin mice challenged for 24 h with HD Lm Δ actA -Ova. (C) Frequency of p65-GFP and TLR9 expression in B cells, CD8a − DC/Mac, CD8a + DCs, plasmacytoid DCs (pDCs), CD103 + DCs, red pulp macrophages (RP Macs), and marginal zone Macs (MZ Macs) (n = 3 per group). (D) Representative ImageStream images displaying TLR9 hi APC subcellular localization of p65-GFP (green) and DAPI (red) and quantitative p65-GFP nuclear translocation (n = 3 per group). (E) C57BL/6 and Tlr9 M7Btlr mice exposed to HD Lm Δ actA -Ova for 24 h. Frequency and absolute number of IL-12 (p40) + and IFN-β + APC subsets after PMA/ionomycin stimulation (n = 4 per group). (F) C57BL/6 and Batf3 −/− mice infected with HD Lm Δ actA -Ova for 24 h. Change in FoxP3 + :FoxP3 − CD4 + T cell ratio at day 1 post-infection relative to the naive state (D FoxP3 + :FoxP3 − ) (n = 7 per group).

Article Snippet: ant-IL-12 (p40) (C17.8) PE , Tonbo Biosciences , Cat#50-7123-U100.

Techniques: Infection, Knock-In, Expressing, Translocation Assay

Journal: Cell reports

Article Title: TLR9 Sensing of Self-DNA Controls Cell-Mediated Immunity to Listeria Infection via Rapid Conversion of Conventional CD4 + T Cells to T reg

doi: 10.1016/j.celrep.2020.01.040

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ant-IL-12 (p40) (C17.8) PE , Tonbo Biosciences , Cat#50-7123-U100.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Gene Expression, Software, Luminex

Journal: Cellular and Molecular Immunology

Article Title: Characterization and allergic role of IL-33-induced neutrophil polarization

doi: 10.1038/cmi.2017.163

Figure Lengend Snippet: Primers used for real-time PCR analysis

Article Snippet: Fluorescein isothiocyanate-conjugated anti-Ly6G (1A8), PE-conjugated anti-IL-1R2 (4E2), PE-conjugated anti-mSiglec F (E50–2440) were purchased from BD Biosciences Pharmingen (San Diego, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Cellular and Molecular Immunology

Article Title: Characterization and allergic role of IL-33-induced neutrophil polarization

doi: 10.1038/cmi.2017.163

Figure Lengend Snippet: IL-33-induced neutrophils express unique cytokine and chemokine receptors compared with N0- or LPS-treated neutrophils. (a) Expression profiling of lineage-restricted receptors of cytokines and chemokines in N0, N(LPS) and N(IL-33) cells. Genes with predominant expression levels in one cell type (at least twofold change compared with other cell types) were organized to draw the plot. Colors represent genes above (red) or below (blue) the second highest expression in three cell types. (b) Freshly isolated bone marrow neutrophils were stimulated with LPS and IL-33 for the indicated times. The levels of cytokine and chemokine receptors in N(IL-33) cells, including IL-13Rα1, CXCR1, CCR1, IL-1R2 and CXCR2, were determined by real-time PCR. (c) Flow cytometric analysis of IL-1R2 expression on the surface of N0, N(LPS) and N(IL-33) cells. (d) The average percentages of IL-1R2+ cells in different neutrophil subpopulations were summarized. Data were shown as mean±s.d. (n=3). *P<0.05, **P<0.01 and ***P<0.001 compared with control or LPS-treated cells. CCR, C-C motif chemokine receptor; CXCR, C-X-C motif chemokine receptor; IL-1R, interleukin-1 receptor; LPS, lipopolysaccharide.

Article Snippet: Fluorescein isothiocyanate-conjugated anti-Ly6G (1A8), PE-conjugated anti-IL-1R2 (4E2), PE-conjugated anti-mSiglec F (E50–2440) were purchased from BD Biosciences Pharmingen (San Diego, CA, USA).

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Immunology

Article Title: Characterization and allergic role of IL-33-induced neutrophil polarization

doi: 10.1038/cmi.2017.163

Figure Lengend Snippet: The specific phenotype of IL-33-treated neutrophils is mediated by JNK and NF-κB signaling pathways. (a) Neutrophils freshly isolated from bone marrow were treated with IL-33 for 15, 30 and 120 min, respectively. The activities of JNK, p38, STAT1, Erk and p65 in cytoplasm were detected by western blotting. (b) The c-jun and p65 protein levels in nucleus of neutrophils after IL-33 treatment for 1 and 2 h, respectively, were determined by western blotting. (c and d) Neutrophils were pretreated with the indicated concentrations of JNK inhibitor (SP600125) and NF-κB inhibitor for 30 min and then stimulated with IL-33 for 24 h. The levels of IL-9, CCL2, CCL7, CCL12, IL-1R2 and IL-13Ra1 expression were detected by real-time PCR. (e and f) The concentrations of IL-9, IL-4, IL-5, IL-13, CCL2 and CCL7 in the supernatant of neutrophils pretreated with either SP600125 or NF-κB inhibitor and then cultured with IL-33 for 24 h were determined by ELISA assay kits. Assays were performed more than three times. Data are shown as mean±s.d. (n=3). *P<0.05, **P<0.01 and ***P<0.001 compared with control. CCL, C-C motif chemokine ligand; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal–regulated kinase; IL, interleukin; JNK, c-Jun N-terminal kinase; NF, nuclear factor; STAT, signal transducer and activator of transcription factor.

Article Snippet: Fluorescein isothiocyanate-conjugated anti-Ly6G (1A8), PE-conjugated anti-IL-1R2 (4E2), PE-conjugated anti-mSiglec F (E50–2440) were purchased from BD Biosciences Pharmingen (San Diego, CA, USA).

Techniques: Isolation, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Immunology

Article Title: Characterization and allergic role of IL-33-induced neutrophil polarization

doi: 10.1038/cmi.2017.163

Figure Lengend Snippet: N(IL-33) neutrophils promote OVA-induced allergic airway inflammation in vivo. OVA-presensitized WT and IL-33-TG mice were challenged with aerosolized OVA daily for 3 days as described in the Materials and methods section. (a) IL-9+ and IL-1R2+ neutrophils in BAL fluid of wild-type asthma mice were detected by flow cytometry. (b) H&E staining of lung tissues from control or OVA-induced asthma WT and IL-33-TG mice is presented. (c) Ratio of wet/dry lung weights of WT and IL-33-TG mice after OVA challenge was measured as described in the Materials and methods section. (d) Cell numbers in BAL fluid of WT and IL-33-TG mice after OVA challenge. (e) Flow cytometric analysis of infiltrated cells such as eosinophils in BAL fluid of WT and IL-33-TG mice after OVA challenge. (f) Proportion of neutrophils in BAL fluid of WT and IL-33-TG mice after OVA challenge is presented. (g) The mRNA expression levels of IL-4, IL-5, IL-9, IL-13, CCL2 and CCL7 in lung tissues of WT and IL-33-TG mice after OVA challenge were detected by real-time PCR. (h) Wild-type neutrophils were stimulated with IL-33, and N0 or N(IL-33) cells were transferred into OVA-presensitized syngeneic wild-type recipients before challenged with aerosolized OVA for 3 days as described in the Materials and methods section. H&E staining of lung tissues from mice treated as described. (i) Cell numbers in BAL fluid of OVA-induced asthma mice transferred with N0 and N(IL-33) cells. (j) The infiltrated cells in BAL fluid of asthma mice treated with N0 and N(IL-33) cells were detected by flow cytometry. Eosinophil numbers in BAL fluid were presented. (k and l) Quantitative PCR analysis of IL-4, IL-5, IL-9, IL-13, CCL2 and CCL7 expression levels in lung tissues of asthma mice treated with N0 and N(IL-33) cells. Data are expressed as mean±s.d. (n=3–5) and are one representative of the three independent experiments with similar results. *P<0.05, **P<0.01 and ***P<0.001 compared with control mice. BAL, bronchoalveolar lavage; CCL, C-C motif chemokine ligand; H&E, hematoylin and eosin; IL, interleukin; OVA, ovalbumin; WT, wild type.

Article Snippet: Fluorescein isothiocyanate-conjugated anti-Ly6G (1A8), PE-conjugated anti-IL-1R2 (4E2), PE-conjugated anti-mSiglec F (E50–2440) were purchased from BD Biosciences Pharmingen (San Diego, CA, USA).

Techniques: In Vivo, Flow Cytometry, Staining, Expressing, Real-time Polymerase Chain Reaction