Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: WT and Tlr9–/– mice were subjected to CLP. PLF was collected at 18 hours after CLP. (A) Percentages of indicated immune cells were measured by flow cytometry. (B) TLR9 expression in indicated peritoneal cells. MFI for TLR9 expression was measured by flow cytometry. (C–G) Peritoneal cell counts of (C) B cells, (D) macrophages, (E) DCs, (F) neutrophils, and (G) T cells in control and after CLP. Data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired, 2-tailed Student’s t tests.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: Flow Cytometry, Expressing

Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: (A–D) WT and Tlr9–/– mice were subjected to CLP. Plasma and PLF were collected at 18 hours after CLP. (A) Peritoneal B-1 cell numbers. (B) Plasma IgM levels. (C) Peritoneal IgM levels. (D) Peritoneal GM-CSF levels. (E–G) Tlr9–/– mice were treated with CD19 neutralizing antibodies (10 mg/mouse) or control IgG (10 mg/mouse) at 24 hours before CLP. PLF was collected at 18 hours after CLP. (E) Seven-day survival after CLP. Data are from 2 separate experiments. n = 10. *P < 0.05, log-rank test. (F) Bacterial clearance in PLF. Data are from 2 separate experiments. Symbols represent individual mice. *P < 0.05, nonparametric Mann-Whitney U test. (G) Peritoneal IgM levels. For A–D and G, data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired, 2-tailed Student’s t test.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: (A) WT and Tlr9–/– mice were subjected to CLP. PLF was collected at 18 hours after CLP. Peritoneal CXCL13 levels were assessed using ELISA. (B–F) WT and Tlr9–/– mice were treated with CXCL13 neutralizing antibodies (10 mg/mouse) or control IgG (10 mg/mouse) immediately after CLP. PLF and plasma were collected at 18 hours after CLP. (B) Peritoneal B-1 cell numbers. (C) Peritoneal IgM levels. (D) Bacterial load in PLF. (E) Plasma IL-6 levels. (F) Seven-day survival. For A–C and E, data are shown as mean ± SD. Symbols represent individual mice. *P < 0.05; **P < 0.01, unpaired, 2-tailed Student’s t tests. For D, symbols represent individual mice. *P < 0.05, nonparametric Mann-Whitney U test. For F, n = 13–19/group as indicated. *P < 0.05 versus Tlr9–/– IgG, log-rank test. (G–K) WT mice were treated with recombinant CXCL13 (10 mg/mouse) or PBS immediately after CLP. (G) Peritoneal B-1 cell number. (H) Peritoneal IgM levels. (I) Bacterial load in PLF. (J) Plasma IL-6 levels. (K) Seven-day survival. For G, H, and J, data are shown as mean ± SD. Symbols represent individual mice. *P < 0.05, unpaired, 2-tailed Student’s t test. For I, symbols represent individual mice. *P < 0.05, nonparametric Mann-Whitney U test. For K, n = 10/group. *P < 0.05, log-rank test.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: (A–C) WT and Tlr9–/– mice were subjected to CLP. PLF and mesenteric adipose tissue were collected at18 hours after CLP. (A and B) Cxcl13 expression in peritoneal cells (A) and mesenteric adipose tissue (B) were assessed using quantitative PCR. Data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05, 2-tailed Student’s t test. (C) Immunofluorescence staining of CXCL13 (red), CD45 (green), CD11c (white), and nucleus (blue) in mesenteric adipose tissue. Scale bar: 5 μm. (D and E) Cxcl13 expression in mouse FRCs. WT and Tlr9–/– FRCs were isolated from mesenteric adipose tissues and expanded ex vivo. FRCs were stimulated with indicated TLR ligands (ODN1585, 5 μM; ODN1826, 5 μM; ODN2395, 5 μM, LPS, 1 μg/mL; PIC, 20 μg/mL) for 18 hours. Cxcl13 expression was assessed using quantitative PCR. Data are shown as mean ± SD from 1 representative experiment. Experiments were performed 3 times. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Bonferroni’s post hoc analysis.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Isolation, Ex Vivo

Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: (A) Schematic timeline of experimental set and analysis for adoptive transfer studies. (B and C) Mice were subjected to CLP. WT or Tlr9–/– FRCs (2 × 105/ mouse) were injected i.p. at 1 hour after CLP. (B) Bacterial load in PLF at 18 hours after CLP. (C) Circulating IL-6 levels at 18 hours after CLP. Data are shown as mean ± SD from 2 separate experiments. *P < 0.05, unpaired, 2-tailed Student’s t test. (D and E) Seven-day survival. (D) Mice were subjected to CLP. PBS, WT, or Tlr9–/– FRCs (2 × 105/ mouse) were injected i.p. 1 hour after CLP. n = 13 in PBS group; n = 19 in WT FRC group; n = 22 in Tlr9–/– FRC group. (E) Mice were subjected to CLP. PBS, WT, or Tlr9–/– FRCs (2 × 105/ mouse) were injected i.p. at 12 hours after CLP. n = 10 in PBS group; n = 14 in WT FRC group; n = 15 in Tlr9–/– FRC group. *P < 0.05 vs. PBS, log-rank test. Data are from 3 separate experiments for D and 2 separate experiments for E. Statistical differences were determined using the log-rank test.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: Adoptive Transfer Assay, Injection

Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: Human FRCs were isolated from adipose tissue and expanded ex vivo. (A) TLR9 expression in human FRCs. TLR9 expression was assessed using flow cytometry. Numbers indicate percentage of FRCs (CD45–CD31–PDPN+). (B) Chemokine expression in human FRCs. FRCs were stimulated with indicated TLR ligands (ODN2216, 5 μM; LPS, 1 μg/mL; PIC, 20 μg/mL) for 18 hours. Indicated chemokine expression was assessed using quantitative PCR. Data are shown as mean ± SD from 1 representative experiment. Experiments were performed 3 times. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Bonferroni’s post hoc analysis.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: Isolation, Ex Vivo, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

doi: 10.1172/JCI127542

Figure Lengend Snippet: Activation of TLR9 signaling in FRCs suppresses the production of chemokines, which are essential for recruiting B cells, T cells, macrophages, and neutrophils into the peritoneal cavity as well as driving the formation of FALCs.

Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

Techniques: Activation Assay

Journal: Immunology

Article Title: The Toll‐like receptor 9 signalling pathway regulates MR 1‐mediated bacterial antigen presentation in B cells

doi: 10.1111/imm.12759

Figure Lengend Snippet: Toll‐like receptor 9 (TLR9) activation increases MR1 surface expression in B cells. (a) B‐LCL cells were treated overnight with a human TLR 1–9 agonist kit and ODN2216 (TLR9), using 10‐fold serial dilutions of the stock solutions as indicated in the Materials and methods section. Fixed Escherichia coli (MOI 300) and 6‐formylpterin (50 μm) were included as positive controls. The cells were stained with an allophycocyanin‐conjugated anti‐MR1 antibody and analysed by flow cytometry. Data from three independent experiments are summarized and the relative mean fluorescent intensity (MFI, Vehicle = 1) is shown. Data from agonist‐treated cells were compared with vehicle‐treated cells. *, P < 0·05. (b) B‐LCL cells were treated with fixed E. coli, the TLR9 agonist CpG ODN2216 or control ODN overnight. The cells were stained with an allophycyanin‐conjugated anti‐MR1 antibody. (c) To determine whether CpG increases the level of total cellular MR1, the cells were fixed, permeabilized and stained with allophycocyanin‐conjugated anti‐MR1. (d) B‐LCL cells were treated overnight with fixed E. coli and stained for surface or total MR1 expression. (e) B‐LCL cells were treated overnight with the TLR9 agonist CpG ODN2216 alone, or in the presence of the TLR9 antagonist ODN TTAGGG. The cells were stained with an allophycocyanin‐conjugated anti‐MR1. (f) Human peripheral blood mononuclear cells were treated with fixed E. coli or the TLR9 agonist CpG ODN2216 overnight. The cells were stained with a phycoerythrin‐Cy7‐conjugated anti‐CD19 and allophycocyanin‐conjugated anti‐MR1 monoclonal antibody. MR1 surface expression on CD19+ peripheral blood mononuclear cells is shown. The data shown are representative of at least three independent experiments.

Article Snippet: A human TLR1–TLR9 agonist kit containing stock solutions of Pam3CSK4 (TLR1/2, 100 μg/ml), HKLM (TLR2, 10 10 cells/ml), Poly(I : C)‐HMW (TLR3, 1 mg/ml), LPS (TLR4, 100 μg/ml), Flagellin (TLR5, 100 μg/ml), FSL1 (TLR6/2, 100 μg/ml), Imiquimod (TLR7, 100 μg/ml), ssRNA40 (TLR8, 100 μg/ml) and ODN2006 (TLR9, 500 μ m ), was purchased from InvivoGen (San Diego, CA).

Techniques: Activation Assay, Expressing, Staining, Flow Cytometry

Journal: Immunology

Article Title: The Toll‐like receptor 9 signalling pathway regulates MR 1‐mediated bacterial antigen presentation in B cells

doi: 10.1111/imm.12759

Figure Lengend Snippet: Toll‐like receptor 9 (TLR9) agonists have different effects on MR1 surface expression in other cell types. (a, b, c) Human peripheral blood mononuclear cells (PBMCs) were treated with 5 μm of CpG‐A or CpG‐B, or fixed Escherichia coli (MOI = 100) overnight (solid line). Vehicle‐treated PBMCs were used as controls (grey filled histogram). The cells were co‐stained with an allophycocyanin‐conjugated anti‐MR1 monoclonal antibody (mAb), AlexaFluor488‐conjugated anti‐human CD3, V450‐conjugated anti‐human CD16 or AlexaFluor700‐conjugated anti‐human CD14 mAbs. MR1 expression on T cells (CD3+‐gated, a), natural killer cells (CD16+‐gated, b) and monocytes (CD14+‐gated, c) is shown. The bar graphs quantify MR1's MFI on each cell type when treated with various concentrations of CpG DNA or fixed E. coli (MOI = 100) as indicated. The MFI of MR1 surface expression on T cells, natural killer cells and monocytes from three different healthy donors is displayed relative to vehicle‐treated cells (vehicle = 1). (d) Human PBMCs and THP‐1 cells were stained with an AlexaFluor700‐conjugated anti‐CD14 and allophycocyanin‐conjugated anti‐MR1 mAb. MR1 expression on CD14+‐gated cells is shown. (e) Human PBMCs and THP‐1 cells were treated with CpG‐A, CpG‐B or fixed E. coli overnight. The cells were stained with an AlexaFluor700‐conjugated anti‐CD14 and allophycocyanin‐conjugated anti‐MR1 mAb. The MFI of MR1 expression is shown. (f) THP‐1 cells were treated with a human TLR1–9 agonist kit and ODN2216 (TLR9), using 10‐fold serial dilutions of the stock solutions as indicated. The cells were stained with an allophycocyanin‐conjugated anti‐MR1 mAb. The data shown are representative of at least two independent experiments. *, P < 0·05; ***, P < 0·001.

Article Snippet: A human TLR1–TLR9 agonist kit containing stock solutions of Pam3CSK4 (TLR1/2, 100 μg/ml), HKLM (TLR2, 10 10 cells/ml), Poly(I : C)‐HMW (TLR3, 1 mg/ml), LPS (TLR4, 100 μg/ml), Flagellin (TLR5, 100 μg/ml), FSL1 (TLR6/2, 100 μg/ml), Imiquimod (TLR7, 100 μg/ml), ssRNA40 (TLR8, 100 μg/ml) and ODN2006 (TLR9, 500 μ m ), was purchased from InvivoGen (San Diego, CA).

Techniques: Expressing, Staining

Journal: Immunology

Article Title: The Toll‐like receptor 9 signalling pathway regulates MR 1‐mediated bacterial antigen presentation in B cells

doi: 10.1111/imm.12759

Figure Lengend Snippet: Toll‐like recptor 9 (TLR9) is required for MR1 surface expression. (a) Western blots to confirm reduced TLR9 in B‐LCL cells expressing TLR9 short hairpin RNA (shRNA) or a negative control (NC). The bands were quantified using imageJ and the data shown in (b). (c) TLR9‐specific shRNA and NC cells were treated with CpG‐A overnight. Surface expression of MR1 is shown in the histograms. The relative MFI (NC = 1) of MR1 is shown in (d). (e) TLR9 shRNA and NC cells were treated with fixed Escherichia coli overnight. Surface expression of MR1 is displayed relative to the control (NC = 1). (f) TLR9 shRNA and NC cells were treated with either 2× concentrated CpG‐A conditioned medium (CM) or CpG‐A overnight. The cells were stained with an allophycocyanin‐conjugated anti‐MR1 antibody. The relative MFI of MR1 compared with the control is shown (NC = 1). The data are representative of at least two independent experiments.

Article Snippet: A human TLR1–TLR9 agonist kit containing stock solutions of Pam3CSK4 (TLR1/2, 100 μg/ml), HKLM (TLR2, 10 10 cells/ml), Poly(I : C)‐HMW (TLR3, 1 mg/ml), LPS (TLR4, 100 μg/ml), Flagellin (TLR5, 100 μg/ml), FSL1 (TLR6/2, 100 μg/ml), Imiquimod (TLR7, 100 μg/ml), ssRNA40 (TLR8, 100 μg/ml) and ODN2006 (TLR9, 500 μ m ), was purchased from InvivoGen (San Diego, CA).

Techniques: Expressing, Western Blot, shRNA, Negative Control, Staining

Journal: Immunology

Article Title: The Toll‐like receptor 9 signalling pathway regulates MR 1‐mediated bacterial antigen presentation in B cells

doi: 10.1111/imm.12759

Figure Lengend Snippet: Reduced bacterial antigen presentation in Toll‐like receptor 9 (TLR9) ‐deficient cells. (a) Human Vα7.2+ CD161+ T (MAIT) cells isolated from human peripheral blood mononuclear cells were sorted by flow cytometry. The cells were co‐cultured with B‐LCL cells in the presence or absence of CpG or fixed Escherichia coli for 3 days. An MR1‐specific or isotype control antibody was added to block MAIT cell activation. Supernatants were harvested and interleron‐γ was measured by ELISA. (b) B‐LCL cells expressing NC and TLR9 short hairpin RNA were treated with fixed E. coli overnight. The cells were fixed and co‐cultured with MAIT cells for 3 days. MAIT cell activation was measured by interferon‐γ production into the supernatants. Each bar is the mean of triplicate samples ±SD. *P < 0·05; **P < 0·01. **** P < 0·0001 The data are representative of at least three independent experiments.

Article Snippet: A human TLR1–TLR9 agonist kit containing stock solutions of Pam3CSK4 (TLR1/2, 100 μg/ml), HKLM (TLR2, 10 10 cells/ml), Poly(I : C)‐HMW (TLR3, 1 mg/ml), LPS (TLR4, 100 μg/ml), Flagellin (TLR5, 100 μg/ml), FSL1 (TLR6/2, 100 μg/ml), Imiquimod (TLR7, 100 μg/ml), ssRNA40 (TLR8, 100 μg/ml) and ODN2006 (TLR9, 500 μ m ), was purchased from InvivoGen (San Diego, CA).

Techniques: Isolation, Flow Cytometry, Cell Culture, Blocking Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, shRNA

Journal: Immunology

Article Title: The Toll‐like receptor 9 signalling pathway regulates MR 1‐mediated bacterial antigen presentation in B cells

doi: 10.1111/imm.12759

Figure Lengend Snippet: Early endosomal Toll‐like receptor 9 (TLR9) signalling by CpG‐A enhances MR1 surface expression and antigen presentation. (a) TRAF3 short hairpin RNA (shRNA) and NC cells were treated with CpG‐A overnight. The cells were stained with an allophycocyanin‐conjugated anti‐MR1 monoclonal antibody (mAb) and analysed by flow cytometry. (b) Interferon regulatory factor 7 (IRF7) shRNA and NC cells were treated with CpG‐A overnight. The cells were stained with an allophycocyanin‐conjugated anti‐MR1 mAb for MR1 cell surface expression analysis by flow cytometry. (c) B‐LCL cells were treated with fixed Escherichia coli in the presence of the indicated concentrations of bafilomycin A, chloroquine or brefeldin A overnight. The cells were stained with an allophycocyanin‐conjugated anti‐MR1 mAb and surface MR1 was analysed by flow cytometry. The relative MFI of MR1 expression compared with the control is shown (vehicle = 100). (d) B‐LCL cells were treated with bafilomycin A1 (Baf) or chloroquine (Chl) overnight, fixed and co‐cultured with MAIT cells in the presence of fixed E. coli for 3 days. (e) B‐LCL cells were treated with brefeldin A (BFA) or monensin (Mon) overnight, fixed, and co‐cultured with MAIT cells in the presence of fixed E. coli for 3 days. Activation of MAIT cells was measured by interferon‐γ production into the supernatants. Each bar is the mean of duplicate samples ±SD. **P < 0·01. Representative data from three independent experiments are shown.

Article Snippet: A human TLR1–TLR9 agonist kit containing stock solutions of Pam3CSK4 (TLR1/2, 100 μg/ml), HKLM (TLR2, 10 10 cells/ml), Poly(I : C)‐HMW (TLR3, 1 mg/ml), LPS (TLR4, 100 μg/ml), Flagellin (TLR5, 100 μg/ml), FSL1 (TLR6/2, 100 μg/ml), Imiquimod (TLR7, 100 μg/ml), ssRNA40 (TLR8, 100 μg/ml) and ODN2006 (TLR9, 500 μ m ), was purchased from InvivoGen (San Diego, CA).

Techniques: Expressing, shRNA, Staining, Flow Cytometry, Cell Culture, Activation Assay

Journal: Immunology

Article Title: The Toll‐like receptor 9 signalling pathway regulates MR 1‐mediated bacterial antigen presentation in B cells

doi: 10.1111/imm.12759

Figure Lengend Snippet: Illustration showing early endosomal Toll‐like receptor 9 (TLR9)‐dependent signalling in the control of MR1‐mediated bacterial antigen presentation in antigen‐presenting cells. Upon a bacterial infection, activation of the TLR9 early endosomal signalling pathway enhances the endocytic trafficking of MR1 to the cell surface and thereby regulates MR1‐mediated bacterial antigen presentation. EE: early endosomes; ER: endoplasmic reticulum. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: A human TLR1–TLR9 agonist kit containing stock solutions of Pam3CSK4 (TLR1/2, 100 μg/ml), HKLM (TLR2, 10 10 cells/ml), Poly(I : C)‐HMW (TLR3, 1 mg/ml), LPS (TLR4, 100 μg/ml), Flagellin (TLR5, 100 μg/ml), FSL1 (TLR6/2, 100 μg/ml), Imiquimod (TLR7, 100 μg/ml), ssRNA40 (TLR8, 100 μg/ml) and ODN2006 (TLR9, 500 μ m ), was purchased from InvivoGen (San Diego, CA).

Techniques: Infection, Activation Assay