Journal: Signal Transduction and Targeted Therapy

Article Title: Analogous comparison unravels heightened antiviral defense and boosted viral infection upon immunosuppression in bat organoids

doi: 10.1038/s41392-022-01247-w

Figure Lengend Snippet: Bat organoids elicited a more robust antiviral response to Poly(I:C) treatment. Bat and human intestinal organoids were sheared mechanically and treated with 10 μg/ml Poly(I:C) or mock-treated with DMSO. a , b Induction of IFNs (left) and ISGs (right) in bat intestinal organoids ( a ) and human intestinal organoids ( b ) at the indicated hours after Poly(I:C) treatment. Results show the log2-fold change of GAPDH-normalized expression level in the treated organoids relative to mock-treated organoids. Data represent the mean and s.d. of a representative experiment in organoids from a bat and human donor, n = 3. c Culture media from the treated or mock-treated human intestinal organoids at the indicated hours were applied to ELISA to measure concentrations of IFNL1 and IFNL3. Data represent the mean and s.d. of a representative experiment in one line of human organoids, n = 3. Two-tailed unpaired Student’s t test. d Poly(I:C) treated or mock-treated human intestinal organoids were collected at the indicated hours post-treatment and subjected to Western blot to detect human ISG15. e , f Bat and human intestinal organoids were sheared and incubated with 10 μg/ml Poly(I:C) Fluorescein in triplicate for 2 h and 6 h. The organoids were then dissociated and applied to flow cytometry to detect the percentage at 2 h post treatment ( e ) and mean fluorescence intensity (MFI, f ) of Fluorescein-positive cells at 2 and 6 h post treatment. g , h Induction of TNF-a and IL6 ( g ) and IP10 ( h ) in bat and human intestinal organoids at the indicated hours after treatment. Results show the fold change of GAPDH-normalized expression level in Poly(I:C)-treated organoids relative to mock-treated organoids. Data represent the mean and s.d. of a representative experiment in organoids from a bat and human donor, n = 3

Article Snippet: Cell-free media were harvested from treated and mock-treated human intestinal organoids for measuring the amount of human IFNL1 and IFNL3 using ELISA kits (R&D Systems, DY7246, D28B00).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Western Blot, Incubation, Flow Cytometry, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Analogous comparison unravels heightened antiviral defense and boosted viral infection upon immunosuppression in bat organoids

doi: 10.1038/s41392-022-01247-w

Figure Lengend Snippet: TLR3 and RLR signaling pathways mediate antiviral responses in bat and human organoids. a A schematic graph outlines the experimental procedure for panels b – g . Bat and human intestinal organoids were pretreated with BX795 (0, 0.1 and 1 μM) or CYT387 (0, 0.1, and 1 μg/ml) overnight. After mechanical shearing, the organoids were incubated with or without 10 μg/ml Poly(I:C), together with the initial concentrations of BX795 or CYT387. At the indicated hours after Poly(I:C) treatment, the organoids were harvested and subjected to RT-qPCR assay to examine mRNA expression levels of bat and human IFNL1 and IFNL3; cell-free media from human organoids were applied to ELISA to detect IFNL1 and IFNL3. Data represent the mean and s.d. of a representative experiment in organoids from a bat and human donor, n = 3. Ordinary one-way ANOVA with Tukey’s multiple comparison test. b , c Induction of IFNL1 (left) and IFNL3 (right) in bat intestinal organoids treated with BX795 ( b ) and CYT387 ( c ). Results show the fold change of GAPDH-normalized expression level in the Poly(I:C)-treated organoids relative to mock-treated organoids. d , e Induction of IFNL1 and IFNL3 in human intestinal organoids treated with BX795 ( d ) and CYT387 ( e ). IFNL1 ( f ) and IFNL3 ( g ) secretion from the human organoids at 4 h post-stimulation. h A schematic graph describes the experimental procedure for panels i and j . Bat and human intestinal organoids were pretreated with 1 μg/ml CYT387 or DMSO overnight. After mechanical shearing, the organoids were incubated with 1 μg/ml CYT387 or DMSO with or without 10 μg/ml Poly(I:C) for 4 h. Cell-free media were then harvested and subjected to PRM-MS to analyze bat IFNL3 ( i ) and ISG15 of bat and human ( j ). The dotted lines represent the detection limit. Data represent the mean and s.d. of a representative experiment in organoids from a bat and human donor, n = 3. Two-tailed unpaired Student’s t test

Article Snippet: Cell-free media were harvested from treated and mock-treated human intestinal organoids for measuring the amount of human IFNL1 and IFNL3 using ELISA kits (R&D Systems, DY7246, D28B00).

Techniques: Protein-Protein interactions, Incubation, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Human Placental Trophoblasts Infected by Listeria monocytogenes Undergo a Pro-Inflammatory Switch Associated With Poor Pregnancy Outcomes

doi: 10.3389/fimmu.2021.709466

Figure Lengend Snippet: Top 50 Induced Genes.

Article Snippet: IFNL1, IFNL2, and IFNL3 ELISAs were performed using the human IL-29 (DY7246), IL-28A (DY1587), and IL-28B (DY5259) DuoSet ELISA kits (R&D Systems) per the manufacturer’s instructions.

Techniques:

Journal: Immunity

Article Title: Human TLR-7-, -8-, and -9-Mediated Induction of IFN-α/β and -λ Is IRAK-4 Dependent and Redundant for Protective Immunity to Viruses

doi: 10.1016/j.immuni.2005.09.016

Figure Lengend Snippet: Production of IFN-α/β, IFN-λ, and TNF-α after Stimulation with Viruses in IRAK-4-Deficient Blood Cells PBMCs from controls and/or from IRAK-4-deficient patients P2, P3, or P7 were left unstimulated or stimulated with various intact and UV-inactivated viruses. (A) IFN-β mRNA levels were analyzed by reverse transcription and real-time quantitative PCR 24 hr after stimulation with intact viruses (see for the multiplicities of infection, moi). Means and standard deviations (SD) were calculated for the controls from three independent individuals, each tested once. The patients were each tested once. (B and C) IFN-α secretion (B) and IFN-λ secretion (C) were measured by ELISA after 24 hr of stimulation. Means and standard deviations (SD) were calculated for the controls from 14 independent individuals, each tested once. (D) IFN-α secretion by control PBMCs was measured by ELISA after 24 hr of stimulation by intact or UV-inactivated viruses. Means and standard deviations (SD) were calculated for the controls from seven independent individuals, each tested once. (E) TNF-α secretion was measured by ELISA after 24 hr of stimulation by two intact viruses. Means and standard deviations (SD) for controls were calculated from two independent individuals.

Article Snippet: Plates were coated with 1 μg/ml of anti-human IFN-λ mAb (AF1598, R&D Systems) overnight at 4°C, and the IFN-λ levels in the supernatant were estimated with a secondary biotinylated anti-human IFN-λ mAb (BAF1598, R&D Systems) used at a concentration of 400 μg/ml ( ).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Infection, Enzyme-linked Immunosorbent Assay, Control

Journal: Immunity

Article Title: Human TLR-7-, -8-, and -9-Mediated Induction of IFN-α/β and -λ Is IRAK-4 Dependent and Redundant for Protective Immunity to Viruses

doi: 10.1016/j.immuni.2005.09.016

Figure Lengend Snippet: IFN-β, IFN-λ, and IL-6 Induction in IRAK-4-Deficient Fibroblasts upon Stimulation by Poly(I:C), IL-1β, TNF-α, and Viruses Induction of IFN-β, IFN-λ, and IL-6 in response to poly(I:C) (50 μg/ml), IL-1β (20 ng/ml), TNF-α (10 ng/ml), and viral stimulations in IRAK-4-deficient (P1, P2, or P4) and control fibroblasts. (A and B) IFN-β mRNA induction, as determined by Q-PCR 2 hr after stimulation (A) and IFN-β, as determined by ELISA 24 hr after stimulation (B). Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice. (C) IFN-β and IFN-λ as determined by ELISA 24 hr after stimulation by poly(I:C), following prior treatment with IFN-α2b (10 5 U/ml for 12 hr). This experiment is representative of two independent experiments. (D) IL-6, as determined by ELISA 24 hr after stimulation. Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice. (E) IFN-β mRNA induction 24 hr after stimulation by seven intact viruses (see for the multiplicities of infection, moi). Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice.

Article Snippet: Plates were coated with 1 μg/ml of anti-human IFN-λ mAb (AF1598, R&D Systems) overnight at 4°C, and the IFN-λ levels in the supernatant were estimated with a secondary biotinylated anti-human IFN-λ mAb (BAF1598, R&D Systems) used at a concentration of 400 μg/ml ( ).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Infection