Journal: Journal of Clinical Immunology

Article Title: Suppression of Type I Interferon Signaling in Myeloid Cells by Autoantibodies in Severe COVID-19 Patients

doi: 10.1007/s10875-024-01708-7

Figure Lengend Snippet: Detection of neutralizing auto-abs to IFN-α2, IFN-β, and IFN-ω in severe or critical COVID-19 patients. a . Measurement of auto-abs to human IFN-α2 in the plasma from mild, severe and critical COVID-19 patients using an ELISA. Black or gray circles, diamond, triangles, and rectangles represent patients carrying anti-IFN-α2 antibodies ( n =7). b . Neutralizing activity of the auto-abs to IFN-α2. The phosphorylation of STAT1 in U937 cells stimulated with recombinant human IFN-α2 was evaluated in the presence of 10% plasma from patients ( n =123). Black or gray circles, diamond, triangles, and rectangles represent patients carrying neutralizing anti-IFN-α2 antibodies ( n =7). c. Neutralizing activity of auto-abs to IFN-β or IFN-ω. Phosphorylation of STAT1 in U937 cells stimulated with recombinant human IFN-β or IFN-ω was evaluated in the presence of 10% plasma from patients ( n =123). d . Characteristics of each patient with neutralizing auto-abs to type I IFNs

Article Snippet: The U937 cells were adjusted to 5 × 10 4 cells/40 μl and mixed with 5 μl of plasma (1:10 dilution), rhIFN-α2 (11101-1; R&D), rhIFN-β (8499-IF; R&D), or rhIFN-ω (11395-1; R&D) (final concentration 10 ng/ml).

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Recombinant

Journal: Journal of Neuroinflammation

Article Title: Type I/type III IFN and related factors regulate JEV infection and BBB endothelial integrity

doi: 10.1186/s12974-023-02891-x

Figure Lengend Snippet: The role of IFNs in IFIT induction and hBMEC barrier maintenance. hBMECs were infected with JEV P3, the cells/cell culture supernatants were harvested at 0, 12, 36, and 72 hpi, and the expression of IFN-β/IFN-λ1 at the mRNA and protein levels was determined by utilizing RT-qPCR ( A , B ) and ELISA ( C , D ). E , F hBMECs were treated with 10 ng/ml rhIFN-β ( E ) or 100 ng/ml rhIFN-λ1 protein ( F ) for different time points, and IFIT expression was measured by Western blotting. G , H HEK-293T cells were transfected with either empty vector plasmid (vector) or IFN-β (Vector-IFN-β)/IFN-λ1 (Vector-IFN-λ1) expression plasmid as indicated, and IFIT expression was measured by performing RT-qPCR ( G ) and Western blotting ( H ). I , J Vero cells were treated with poly I: C or rhIFN-β/rhIFN-λ1 protein as indicated, and IFIT expression was measured by performing RT-qPCR ( I ) and Western blotting ( J ). K HEK-293T cells were transfected with either empty vector plasmid (vector) or plasmids expressing IFIT1 (Vector-IFIT1), IFIT2 (Vector-IFIT2), IFIT3 (Vector-IFIT3), and IFIT5 (Vector-IFIT5) as indicated, and IFN-β and IFN-λ1 mRNA expression was measured by performing RT-qPCR. (L) Dual-luciferase reporter assays detected IRF1 binding to the IFN-β promotor in IFIT-overexpressing HEK-293T cells. HEK-293T cells were transfected with vector-IFIT1, vector-IFIT2, vector-IFIT3, vector-IFIT5, or empty vector plasmid to generate overexpressed cell lines. Simultaneously, empty vector plasmid or vector-IRF1 and IFN-β-Luc alone with pRL-TK plasmids were cotransfected into corresponding overexpressed cell lines. M hBMECs were cultured in the upper chamber of the transwell plate and pretreated with rhIFN-β and rhIFN-λ1 protein, followed by treatment with mock-CM or JEV-CM, and then the TEER (upper graph) was determined at the indicated times. Data are represented as the mean values ± SEMs from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant

Article Snippet: Human IFN-β recombinant protein (rhIFN-β) and human IFN-λ1 recombinant protein (rhIFN-λ1) were purchased from Abbkine (Wuhan, China) and MedChemExpress (Shanghai, China).

Techniques: Infection, Cell Culture, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Plasmid Preparation, Luciferase, Binding Assay

Journal: Journal of Neuroinflammation

Article Title: Type I/type III IFN and related factors regulate JEV infection and BBB endothelial integrity

doi: 10.1186/s12974-023-02891-x

Figure Lengend Snippet: The role of the JAK/STAT pathway in IFIT induction. A , B hBMECs were infected with JEV P3 for different time points, and the mRNA of STAT1 ( A ) and the protein of JAK1/p-JAK1, JAK2/p-JAK2, and STAT1/p-STAT1 ( B ) were measured. C The protein expression of JAK1/p-JAK1, JAK2/p-JAK2, and STAT1/p-STAT1 was measured in PRR-knockdown hBMECs under JEV infection. D , E IFN-β and IFN-λ1 mRNA levels were measured in PRR-knockdown hBMECs under JEV infection. F , G hBMECs were treated with rhIFN-β or rhIFN-λ1 protein for different time points, and JAK1/p-JAK1, JAK2/p-JAK2, and STAT1/p-STAT1 protein expression was measured. H hBMECs were treated with the carrier control DMSO or JAK inhibitor I at various concentrations for 72 h, and cell cytotoxicity was analyzed. I , J hBMECs were pretreated with carrier control DMSO or 5 μM JAK inhibitor I, followed by JEV P3 infection, and the mRNA of STAT1 ( I ) and the protein of JAK1/p-JAK1, JAK2/p-JAK2, and STAT1/p-STAT1 ( J ) were determined. K , L hBMECs were pretreated with the carrier control DMSO or 5 μM JAK inhibitor I, followed by JEV P3 infection, and IFIT mRNA ( K ) and protein ( L ) expression was measured. Data are represented as the mean values ± SEMs from three independent experiments. *** p < 0.001; **** p < 0.0001; ns: not significant

Article Snippet: Human IFN-β recombinant protein (rhIFN-β) and human IFN-λ1 recombinant protein (rhIFN-λ1) were purchased from Abbkine (Wuhan, China) and MedChemExpress (Shanghai, China).

Techniques: Infection, Expressing, Knockdown, Control

Journal: Journal of Neuroinflammation

Article Title: Type I/type III IFN and related factors regulate JEV infection and BBB endothelial integrity

doi: 10.1186/s12974-023-02891-x

Figure Lengend Snippet: IFITs are nonessential in hBMEC barrier integrity. A , B The expression of IFITs was assessed by RT-qPCR (A) and Western blotting ( B ) in IFIT-knockdown hBMECs under JEV infection. C After serum-free starvation, cell viability was determined in IFIT-knockdown hBMECs. D , E IFIT-knockdown hBMECs were seeded onto the upper chamber of the Transwell plate until confluent. At the indicated times, the TEER (upper graph) was measured without treatment ( D ) or with rhIFN-β and rhIFN-λ1 protein treatment prior to the addition of JEV-CM ( E ). Data are represented as the mean values ± SEMs from three independent experiments. ** p < 0.01; **** p < 0.0001

Article Snippet: Human IFN-β recombinant protein (rhIFN-β) and human IFN-λ1 recombinant protein (rhIFN-λ1) were purchased from Abbkine (Wuhan, China) and MedChemExpress (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Infection