Journal: Cell Reports

Article Title: Aryl Hydrocarbon Receptor Contributes to the Transcriptional Program of IL-10-Producing Regulatory B Cells

doi: 10.1016/j.celrep.2019.10.018

Figure Lengend Snippet:

Article Snippet: Negative CD43- Isolation Kit , Miltenyi Biotec , Cat# 130-049-801.

Techniques: Western Blot, Recombinant, Methylation, Adjuvant, Enzyme-linked Immunosorbent Assay, Isolation, cDNA Synthesis, SYBR Green Assay, DNA Library Preparation, Purification, Bicinchoninic Acid Protein Assay, Microarray, Ex Vivo, Generated, Software, Red Blood Cell Lysis, Staining, Lysis

Journal: iScience

Article Title: Sirtuin 2 regulates neutrophil functions through NAD + synthesis pathway in virus infection

doi: 10.1016/j.isci.2024.110184

Figure Lengend Snippet: SIRT2 expression is associated with NAD + synthesis and neutrophil function during virus infection (A) C57BL/6 mice were infected with or without the PR8 virus for 48 h, after which the lungs were collected. RNA was analyzed via microarray analysis to compare the expression profiles of control and virus-infected cells from the lung with those of certain genes involved in surface and intracellular signaling pathways. (B) The expression of the indicated molecules was examined via qPCR analyses of neutrophils in BALF from PBS (ctrl)-treated or virus-infected mice (the expression in the control groups was set to 1). (C) Expressions of SIRT2 and NAMPT in neutrophils in BALF from PBS (ctrl)- or virus (virus)-infected mice by flow cytometry. (D) Sirt2 mRNA expression was examined by qPCR analyses in the indicated cell populations isolated from BALF from virus-infected mice. (E) Sirt2 and Nampt mRNA expression in neutrophils isolated from BALF was examined via qPCR at different time points following virus infection in mice. (F and G) Changes in the NAD + (F) and Qprt mRNA levels (G) in neutrophils isolated from BALF at different time points following virus infection. (H and I) The correlation between the percent of TNF-α + cells among neutrophils (H) or the dry/wet weight ratio of the lung (I) and the expression of Sirt2 in neutrophils during virus infection. The data are representative of three or four independent experiments ( n = 4–10 mice per group). ∗∗ p < 0.01 and ∗∗∗ p < 0.001, compared with the indicated groups.

Article Snippet: SIRT2 deficiency inhibits neutrophil functions through the QA-QPRT-NAD + synthesis pathway (A–F) WT mice were challenged with the PR8 virus at 48 h and treated with PBS (vehicle), AGK2 (5 mg/kg, Selleck), AGK2+FK866 (10 mg/kg, Selleck) or AGK2+FK866+QA (10 mg/kg, Selleck) (A). (B) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with the PR8 virus. (C) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with the PR8 virus. (D) Flow cytometry analysis of CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (E) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (F–I) Human CD34 + hematopoietic stem cells were induced with G-CSF (10 ng/mL, Sigma) for 7 days.

Techniques: Expressing, Virus, Infection, Microarray, Control, Protein-Protein interactions, Flow Cytometry, Isolation

Journal: iScience

Article Title: Sirtuin 2 regulates neutrophil functions through NAD + synthesis pathway in virus infection

doi: 10.1016/j.isci.2024.110184

Figure Lengend Snippet: SIRT2 deficiency limits neutrophil infiltration and function in ameliorating viral infection-related inflammation WT and Sirt2 −/− mice were challenged with PBS (ctrl) or subjected to PR8 virus infection for 48 h. Clinical score of infected mice (A) and the ratio of dry and wet weights of lungs (B) from mice. (C) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with PBS or the PR8 virus. (D) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with PBS or the PR8 virus. (E) Flow cytometry analysis of CD11b + Ly6G + cells in the bronchoalveolar lavage fluid (BALF) or lungs of WT and Sirt2 −/− mice challenged with PBS (ctrl) or PR8 virus infection for 48 h. Dot plots present representative flow cytometry data. (F) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT and Sirt2 −/− mice challenged with PBS (ctrl) or PR8 virus infection for 48 h. Dot plots present representative flow cytometry data. (G) NETs from CD11b + Ly6G + neutrophils were isolated from BALF from WT and Sirt2 −/− mice at 48 h after PR8 infection. Typical NET images are displayed. Scale bars, 10 μm; original magnification, 630×. (H) Intracellular staining of CitH3 in Ly6G + cells in BALF from WT and Sirt2 −/− mice at 48 h after PR8 infection. Dot plots present representative flow cytometry data (upper panel), and the statistical results are shown (lower panel). (I) Western blot analysis of the CitH3 in Ly6G + cells sorted from BALF from WT and Sirt2 −/− mice 48 h after challenge with PBS (ctrl) or PR8 infection. The data are representative of three or four independent experiments ( n = 3–6 mice per group). ∗∗∗ p < 0.001, compared with the indicated groups.

Article Snippet: SIRT2 deficiency inhibits neutrophil functions through the QA-QPRT-NAD + synthesis pathway (A–F) WT mice were challenged with the PR8 virus at 48 h and treated with PBS (vehicle), AGK2 (5 mg/kg, Selleck), AGK2+FK866 (10 mg/kg, Selleck) or AGK2+FK866+QA (10 mg/kg, Selleck) (A). (B) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with the PR8 virus. (C) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with the PR8 virus. (D) Flow cytometry analysis of CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (E) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (F–I) Human CD34 + hematopoietic stem cells were induced with G-CSF (10 ng/mL, Sigma) for 7 days.

Techniques: Infection, Virus, Staining, Immunohistochemical staining, Flow Cytometry, Isolation, Western Blot

Journal: iScience

Article Title: Sirtuin 2 regulates neutrophil functions through NAD + synthesis pathway in virus infection

doi: 10.1016/j.isci.2024.110184

Figure Lengend Snippet: CXCR2 is critically involved in regulating neutrophil function induced by Sirt2 −/− during virus infection Percent (A) and mean fluorescent intensity (MFI; B) of CXCR2 surface expressions among CD11b + Ly6G + neutrophils were isolated from bronchoalveolar lavage fluid (BALF) of WT and Sirt2 −/− mice at 48 h after challenge with PBS (ctrl) or PR8 infection. Representative flow cytometry data (left) and statistical results are shown (right). (C–J) WT and Sirt2 −/− mice were challenged with PBS (ctrl) or PR8 virus infection for 48 h with a CXCR2 inhibitor (CXCR2i; SB265610, 5 mg/kg, intraperitoneal injected daily; Tocris Biosciences) or vehicle (PBS). Clinical score of infected mice (C) and the ratio of dry and wet weights of lungs (D) from mice. Percent of CD11b + Ly6G + cells in the BALF (E) and lungs (F) of mice. Intracellular staining of TNF-α in Ly6G + cells in the BALF (G) and lungs (H) of mice. (I) NETs from CD11b + Ly6G + neutrophils were isolated from the BALF of mice. The percent of NET + cells were quantified. (J) Intracellular staining of CitH3 in Ly6G + cells in BALF from mice. The data are representative of three or four independent experiments ( n = 3–4 mice per group). ∗∗ p < 0.01 and ∗∗∗ p < 0.001, compared with the indicated groups.

Article Snippet: SIRT2 deficiency inhibits neutrophil functions through the QA-QPRT-NAD + synthesis pathway (A–F) WT mice were challenged with the PR8 virus at 48 h and treated with PBS (vehicle), AGK2 (5 mg/kg, Selleck), AGK2+FK866 (10 mg/kg, Selleck) or AGK2+FK866+QA (10 mg/kg, Selleck) (A). (B) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with the PR8 virus. (C) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with the PR8 virus. (D) Flow cytometry analysis of CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (E) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (F–I) Human CD34 + hematopoietic stem cells were induced with G-CSF (10 ng/mL, Sigma) for 7 days.

Techniques: Virus, Infection, Isolation, Flow Cytometry, Injection, Staining

Journal: iScience

Article Title: Sirtuin 2 regulates neutrophil functions through NAD + synthesis pathway in virus infection

doi: 10.1016/j.isci.2024.110184

Figure Lengend Snippet: Epithelial cells help neutrophils use QA for NAD + synthesis induced by Sirt2 −/− following virus infection (A) TDO and 3-HA in the NAD + synthesis pathway. (B) Single-cell suspension was prepared from lung tissue. Mass spectrometry analysis of the metabolite abundance of 3-HA in neutrophils from BALF and in lung tissue from WT and Sirt2 −/− mice challenged with the PR8 virus at 48 h. (C) Mass spectrometry analysis of the metabolite abundance of 3-HA in Epcam1 + and Epicam1 - cells from lung tissue from WT and Sirt2 −/− mice challenged by PR8 virus infection at 48 h. (D) Flow cytometry analysis of TDO expression in neutrophils and epithelial cells in the lungs of WT and Sirt2 −/− mice challenged with the PR8 virus at 48 h. (E) Ido1/2 and Tdo mRNA expression of neutrophils or cells from lung tissue from WT and Sirt2 −/− mice challenged by PR8 virus infection at 48 h. (F–H) Neutrophils were isolated from the spleens of WT and Sirt2 −/− mice cocultured with vehicle (in PBS), epithelial cells or epithelial cells treated with a TDO inhibitor (LM10, 10 nM, MCE) and stimulated with virus for different durations. Ratio of the NAD + level in Sirt2 −/− and WT mice (G) and the ratio of TNF-α + cells in neutrophils from Sirt2 −/− and WT mice (H). The data are representative of three or four independent experiments ( n = 3–4 mice per group). ∗∗ p < 0.01 and ∗∗∗ p < 0.001, compared with the indicated groups. n.s., not significant.

Article Snippet: SIRT2 deficiency inhibits neutrophil functions through the QA-QPRT-NAD + synthesis pathway (A–F) WT mice were challenged with the PR8 virus at 48 h and treated with PBS (vehicle), AGK2 (5 mg/kg, Selleck), AGK2+FK866 (10 mg/kg, Selleck) or AGK2+FK866+QA (10 mg/kg, Selleck) (A). (B) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with the PR8 virus. (C) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with the PR8 virus. (D) Flow cytometry analysis of CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (E) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (F–I) Human CD34 + hematopoietic stem cells were induced with G-CSF (10 ng/mL, Sigma) for 7 days.

Techniques: Virus, Infection, Suspension, Mass Spectrometry, Flow Cytometry, Expressing, Isolation

Journal: iScience

Article Title: Sirtuin 2 regulates neutrophil functions through NAD + synthesis pathway in virus infection

doi: 10.1016/j.isci.2024.110184

Figure Lengend Snippet: SIRT2 deficiency inhibits neutrophil functions through the QA-QPRT-NAD + synthesis pathway (A–F) WT mice were challenged with the PR8 virus at 48 h and treated with PBS (vehicle), AGK2 (5 mg/kg, Selleck), AGK2+FK866 (10 mg/kg, Selleck) or AGK2+FK866+QA (10 mg/kg, Selleck) (A). (B) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with the PR8 virus. (C) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with the PR8 virus. (D) Flow cytometry analysis of CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (E) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (F–I) Human CD34 + hematopoietic stem cells were induced with G-CSF (10 ng/mL, Sigma) for 7 days. Neutrophils were treated with AGK2 (0–10 μM, Selleck) at the indicated dose and stimulated with virus for 24 h (F). (G) Sirt2 mRNA expression in neutrophils. (H) TNF-α production in the supernatant, as determined by ELISA. (I) Qprt and Nampt mRNA expression in neutrophils. The data are representative of three or four independent experiments ( n = 3 per group). ∗ p < 0.05 and ∗∗∗ p < 0.001, compared with the indicated groups.

Article Snippet: SIRT2 deficiency inhibits neutrophil functions through the QA-QPRT-NAD + synthesis pathway (A–F) WT mice were challenged with the PR8 virus at 48 h and treated with PBS (vehicle), AGK2 (5 mg/kg, Selleck), AGK2+FK866 (10 mg/kg, Selleck) or AGK2+FK866+QA (10 mg/kg, Selleck) (A). (B) Hematoxylin and eosin (H&E) staining of mouse lungs at 48 h after challenge with the PR8 virus. (C) Immunohistochemical staining of anti-Ly6G in the lungs at 48 h after challenge with the PR8 virus. (D) Flow cytometry analysis of CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (E) Intracellular staining of TNF-α in CD11b + Ly6G + cells in the BALF or lungs of WT mice challenged with the PR8 virus at 48 h. Dot plots present representative flow cytometry data (left), and the statistical results are shown (right). (F–I) Human CD34 + hematopoietic stem cells were induced with G-CSF (10 ng/mL, Sigma) for 7 days.

Techniques: Virus, Staining, Immunohistochemical staining, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay