Journal: iScience

Article Title: The TLR4–TRIF–type 1 IFN–IFN-γ pathway is crucial for gastric MALT lymphoma formation after Helicobacter suis infection

doi: 10.1016/j.isci.2021.103064

Figure Lengend Snippet: Activation of the TLR4 signaling pathway after H. suis infection (A) Tlr4 mRNA levels in the stomachs of H. suis -infected WT, Myd88 KO, Trif KO, and Myd88 / Trif DKO mice 6 months after infection were determined with qRT-PCR. (B) Immunohistology of TLR4-expressing cells analyzed with confocal microscopy. Magnification, 100×. (C) Immunohistology of EpCAM-positive cells in the stomachs of H. suis -infected WT mice. Magnification, 100×. Formation of gastric lymphoid follicles is indicated by dotted lines. Representative images are shown. (D – H) C57BL/6J WT mice were infected with H. suis for 6 months and gastric cells were analyzed with flow cytometry. (D) Staining with B220 and CD19 antibodies, followed by staining for TLR4. Right panels show TLR4 staining among B220 + CD19 + cells. (E) Staining with CD4 and TCRβ antibodies, followed by staining for TLR4. Right panels show TLR4 staining among CD4+ TCRβ+ cells. (F) Staining for CD11c and MHC class antibodies, followed by staining for TLR4. Right panels show TLR4 staining among CD11c + MHC class + cells.(G) Staining for FDC M1, followed by staining for TLR4. Right panels show TLR4 staining among FDC M1+ cells. (H) Staining for EpCAM, followed by staining for TLR4. Right panels show TLR4 staining among EpCAM + cells. Ifna (I) and Ifnb (J) mRNA levels in the stomach after H. suis infection were determined with qRT-PCR. (A, I, and J) Data were normalized to β-actin levels. Data are shown as the mean ± SD (n = 5) of three independent experiments. ∗∗p < 0.01, N.S., no significant difference (ANOVA). Ab, antibody; DKO, double knockout. Scale bar, 100 μm.

Article Snippet: AGS cells were stimulated with recombinant human IFN-alpha A/D (11,200-2; R&D Systems, Minneapolis, MN, USA) at 9,000 U/mL, recombinant human IFN-beta 1a (11,410-2; R&D Systems) at 9,000 U/mL, recombinant human IFN-γ (285-IF-100; R&D Systems) at 1 μg/mL, TLR4 inhibitor peptide (NBP2-26244; Novus Biologicals, Centennial, CO, USA) at 30 μM, control peptide (NBP2-26244; Novus Biologicals) at 30 μM, and MPLA-SM (tlrl-mpla; Invitrogen, Carlsbad, CA, USA) at 10 μg/mL for 24 or 48 hr.

Techniques: Activation Assay, Infection, Quantitative RT-PCR, Expressing, Confocal Microscopy, Flow Cytometry, Staining, Double Knockout

Journal: iScience

Article Title: The TLR4–TRIF–type 1 IFN–IFN-γ pathway is crucial for gastric MALT lymphoma formation after Helicobacter suis infection

doi: 10.1016/j.isci.2021.103064

Figure Lengend Snippet: Activation of TLR4 signaling in gastric epithelial cells after treatment with monophosphoryl lipid A (MPLA) Gastric epithelial cell lines (AGS cells) were treated with 1 μg/mL MPLA for 24 and 48 hr with or without the TLR4 inhibitor peptide (30 μM) 2 hr before MPLA treatment. (A and B) qRT-PCR analysis of TLR4 mRNA levels in AGS cells after treatment with MPLA at 24 and 48 hr (A) and with or without the TLR4 inhibitor at 48 hr (B). (C–E) (C) qRT-PCR analysis of TRIF mRNA levels in AGS cells after treatment with MPLA, with or without the TLR4 inhibitor, after 48 hr qRT-PCR analysis of IFNA (D) and IFNB (E) expression levels in AGS cells at 24 and 48 hr after MPLA treatment. ELISA of IFN-α (F) and IFN-β (G) production in AGS cells at 24 and 48 hr after MPLA treatment. qRT-PCR analysis of IFNA (H) and IFNB (I) mRNA levels at 48 hr after MPLA treatment with or without the TLR4 inhibitor. Data are shown as the mean ± SD (n = 5) of three independent experiments. qRT-PCR data were normalized to β-actin levels ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, N.S., no significant difference (ANOVA).

Article Snippet: AGS cells were stimulated with recombinant human IFN-alpha A/D (11,200-2; R&D Systems, Minneapolis, MN, USA) at 9,000 U/mL, recombinant human IFN-beta 1a (11,410-2; R&D Systems) at 9,000 U/mL, recombinant human IFN-γ (285-IF-100; R&D Systems) at 1 μg/mL, TLR4 inhibitor peptide (NBP2-26244; Novus Biologicals, Centennial, CO, USA) at 30 μM, control peptide (NBP2-26244; Novus Biologicals) at 30 μM, and MPLA-SM (tlrl-mpla; Invitrogen, Carlsbad, CA, USA) at 10 μg/mL for 24 or 48 hr.

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: The TLR4–TRIF–type 1 IFN–IFN-γ pathway is crucial for gastric MALT lymphoma formation after Helicobacter suis infection

doi: 10.1016/j.isci.2021.103064

Figure Lengend Snippet:

Article Snippet: AGS cells were stimulated with recombinant human IFN-alpha A/D (11,200-2; R&D Systems, Minneapolis, MN, USA) at 9,000 U/mL, recombinant human IFN-beta 1a (11,410-2; R&D Systems) at 9,000 U/mL, recombinant human IFN-γ (285-IF-100; R&D Systems) at 1 μg/mL, TLR4 inhibitor peptide (NBP2-26244; Novus Biologicals, Centennial, CO, USA) at 30 μM, control peptide (NBP2-26244; Novus Biologicals) at 30 μM, and MPLA-SM (tlrl-mpla; Invitrogen, Carlsbad, CA, USA) at 10 μg/mL for 24 or 48 hr.

Techniques: Purification, Control, Virus, Isolation, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Protein Extraction, Software, Real-time Polymerase Chain Reaction, Microplate Reader Absorbance Measurement

Journal: Communications Biology

Article Title: Intrinsic cardiac adrenergic cells contribute to LPS-induced myocardial dysfunction

doi: 10.1038/s42003-022-03007-6

Figure Lengend Snippet: a Co-immunofluorescent staining of TLR4 and TH in NRVM ICA cell+ , TLR4: toll-like receptor 4, red; TH: ICA cells, green; DAPI: nuclei, blue ( n = 6). b and c NE production as well as TH and DBH expression in rat ICA cells treated with Viper (TLR4 inhibitor, 10 μM) for 2 h prior to LPS for 6 h. ( n = 48, each dot represents one independent repeat). d TLR4 expression in wildtype (WT) and TLR4-deficient ( Tlr4 Lps-del ) NMVM ICA cell+ . e and f NE production and DBH expression in WT and Tlr4 Lps-del NMVM ICA cell+ stimulated with LPS for 6 h, control: saline ( n = 12, each dot represents a biological replicate in d – f ). g Luciferase activity of AP-1-luc- and TH-promoter-luc- relative to RLTK-luc- in HEK293/hTLR4 cells at 36 h after transfection and LPS stimulation. h Luciferase activity of AP-1-luc- and TH-promoter-luc- relative to RLTK-luc- in HEK293/hTLR4 cells at 24 h after transfection with plasmids (0.1 μg/mL) encoding MyD88 and TRIF. i Expression of c-Jun and c-Fos in HEK293/hTLR4 cells at 24 h after transfection with MyD88 plasmid, c-Jun siRNA and c-Fos siRNA. j Luciferase activity of TH-promoter-luc- relative to RLTK-luc- in HEK293/hTLR4 cells at 24 h after transfection with MyD88 plasmid, c-Jun siRNA, and c-Fos siRNA, (0.1 μg/mL for plasmid) (each dot represents one independent repeat in ( g – j )). Data are presented as mean ± SEM using one-way ANOVA and Bonferroni multiple comparison test, * P < 0.05, ** P < 0.01, most of the P -values were assigned on the figure. n represents the number of neonatal rats ( a – c ) or neonatal mice ( d – f ).

Article Snippet: α 1 -AR antagonist: Prazosin (Sigma-Aldrich#7791); α 2 -AR antagonist: Yohimbine (Sigma-Aldrich #Y3125); α 2A -AR antagonist: BRL 44408 (Sigma-Aldrich #B4559); β 1 -AR antagonist: CGP20712A (Sigma-Aldrich #C231); β 2 -AR antagonist: ICI-118 551 (Sigma-Aldrich #I127); β 3 -AR antagonist: SR59230A (Sigma-Aldrich #S8688); TLR4 inhibitor: Viper peptide (Novus#NBP2-226244); CaMKII inhibitor: KN-93 Phosphate (Selleckchem #S7423); DBH inhibitor: Nepicastat (MCE MedChem Express#HY-13289); PKA Inhibitor: 14-22 Amide (Calbiochem® #476485).

Techniques: Staining, Expressing, Control, Saline, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Comparison

Journal: Communications Biology

Article Title: Intrinsic cardiac adrenergic cells contribute to LPS-induced myocardial dysfunction

doi: 10.1038/s42003-022-03007-6

Figure Lengend Snippet: ICA cell produces no TNF-α due to the elevated Nfkbia and Tnfaip6 expression. Increased NE release by LPS-TLR4 signaling activates β1-AR-CaMKII signaling in cardiomyocytes to regulate NF-κB and mitogen-activated protein kinase pathways, subsequently aggravating myocardial TNF-α production and dysfunction.

Article Snippet: α 1 -AR antagonist: Prazosin (Sigma-Aldrich#7791); α 2 -AR antagonist: Yohimbine (Sigma-Aldrich #Y3125); α 2A -AR antagonist: BRL 44408 (Sigma-Aldrich #B4559); β 1 -AR antagonist: CGP20712A (Sigma-Aldrich #C231); β 2 -AR antagonist: ICI-118 551 (Sigma-Aldrich #I127); β 3 -AR antagonist: SR59230A (Sigma-Aldrich #S8688); TLR4 inhibitor: Viper peptide (Novus#NBP2-226244); CaMKII inhibitor: KN-93 Phosphate (Selleckchem #S7423); DBH inhibitor: Nepicastat (MCE MedChem Express#HY-13289); PKA Inhibitor: 14-22 Amide (Calbiochem® #476485).

Techniques: Expressing