Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-223 Promotes Type I Interferon Production in Antiviral Innate Immunity by Targeting Forkhead Box Protein O3 (FOXO3) *

doi: 10.1074/jbc.M115.700252

Figure Lengend Snippet: VSV-triggered type I IFN induces miR-223 expression in macrophages. A, RAW264.7 cells were infected with or without VSV at m.o.i. 0.1 for 12 h. Mouse peritoneal macrophages were infected with or without VSV at m.o.i. 10 for 24 h. The expressions of different miRNAs were measured by qPCR and normalized to the expression of U6 in each sample. B, RAW264.7 cells were infected with or without VSV at m.o.i. 0.1 for indicated times or at indicated m.o.i. for 12 h, and the expression of miR-223 was measured by qPCR and normalized to the expression of U6 in each sample. C, mouse peritoneal macrophages were infected with or without VSV at m.o.i. 10 for indicated times or at indicated m.o.i. for 24 h, and the expression of miR-223 was measured. D, RAW264.7 cells were infected with VSV at m.o.i. 0.1, and mouse peritoneal macrophages were infected VSV at m.o.i. 10. Total protein levels of VSV-G in lysates were detected by immunoblot at the indicated time. E, RAW264.7 were treated with suramin (200 μm) after infection with VSV at m.o.i. 0.1 for 1 h and then infected with VSV for 12 h; the expression of VSV RNA replicates, IFN-β and miR-223, were measured by qPCR, and total protein levels of VSV-G in lysates were detected by immunoblot. F, mouse peritoneal macrophages were treated with suramin (200 μm) after infection with VSV at m.o.i. 10 for 1 h and then infected with VSV for 24 h; the expression of VSV RNA replicates, IFN-β and miR-223, were measured by qPCR, and total protein levels of VSV-G in lysates were detected by immunoblot. G, RAW264.7 cells were infected with infectious VSV, heat- or UV-inactivated VSV at m.o.i. 0.1 for 12 h; IFN-β and miR-223 were measured by qPCR. H, mouse peritoneal macrophages were infected with infectious VSV, heat- or UV-inactivated VSV at m.o.i. 10 for 24 h; IFN-β and miR-223 were measured by qPCR. I, mouse peritoneal macrophages were infected with infectious H1N1, heat- or UV-inactivated H1N1 at m.o.i. 10 for 24 h; IFN-β and miR-223 were measured by qPCR. J, RAW264.7 macrophages were treated with rmIFN-β (25 ng/ml) for indicated times or pretreated with anti-mouse IFNAR1 antibody (10 μg/ml) for 2 h, then infected with VSV at m.o.i. 0.1 for indicated times, and the expression of miR-223 was measured. K, mouse peritoneal macrophages were treated as in E, infected with VSV at m.o.i. 10 for indicated times, and miR-223 expression was measured. Data are the mean ± S.D. (n = 3) of one representative experiment. Similar results were obtained in three independent experiments. ***, p < 0.1; **, p < 0.01; *, p < 0.05; ns, not significant; Ctrl, control.

Article Snippet: Recombinant mouse IFN-β (rmIFN-β) was from Sino Biological (Beijing, China).

Techniques: Expressing, Infection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-223 Promotes Type I Interferon Production in Antiviral Innate Immunity by Targeting Forkhead Box Protein O3 (FOXO3) *

doi: 10.1074/jbc.M115.700252

Figure Lengend Snippet: miR-223 positively regulates VSV-triggered type I IFN production. A, 0.5 ml of 2 × 105 RAW264.7 cells were transfected with control (Ctrl) mimics or miR-223 mimics (left), control inhibitors or miR-223 inhibitors (right) as indicated at a final concentration of 20 nm. After 48 h, miR-223 expression was measured. B, 0.5 ml of 2 × 105 mouse peritoneal macrophages were transfected as described in A, and after 48 h miR-223 expression was measured. C, 0.5 ml of 2 × 105 RAW264.7 cells were transfected as described in A. After 48 h, cells were infected by VSV at m.o.i. 0.1 for indicated times. IFN-α4 (left) and IFN-β (right) mRNA expression were measured by qPCR and normalized to the expression of β-actin in each sample. D, 0.5 ml of 2 × 105 mouse peritoneal macrophages were transfected as described in A. After 48 h, cells were infected by VSV at m.o.i. 10 for indicated times. IFN-α4 and IFN-β mRNA expression were measured by qPCR. IFN-β in supernatants was measured by ELISA. E, 0.5 ml of 2 × 105 peritoneal macrophages were transfected with control mimics or miR-223. After 48 h, cells were infected by VSV at m.o.i. 10 for indicated times. TNF-α and IL-6 mRNA expressions were measured by qPCR. TNF-α and IL-6 in supernatants were measured by ELISA. Data are the mean ± S.D. (n = 3) of one representative experiment. Similar results were obtained in three independent experiments. ***, p < 0.1; **, p < 0.01; *, p < 0.05; ns, not significant.

Article Snippet: Recombinant mouse IFN-β (rmIFN-β) was from Sino Biological (Beijing, China).

Techniques: Transfection, Concentration Assay, Expressing, Infection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-223 Promotes Type I Interferon Production in Antiviral Innate Immunity by Targeting Forkhead Box Protein O3 (FOXO3) *

doi: 10.1074/jbc.M115.700252

Figure Lengend Snippet: Antiviral function of miR-223 is mainly through targeting FOXO3. A and B, murine peritoneal macrophages were transfected with control siRNA and FOXO3 siRNA as indicated. After 48 h, FOXO3, IRF7, IFN-α4, and IFN-β mRNA expressions and FOXO3 protein levels were detected. C, murine peritoneal macrophages were transfected with control (Ctrl), miR-223 mimics, and FOXO3 siRNA, respectively, as indicated. After 48 h, macrophages were infected by VSV at m.o.i. 10 for 1 h and washed; intracellular VSV RNA replicates at indicated time points were quantified using qRT-PCR, and the value at 4-h time point in control cells served as 1-fold control. The relative IFN-β mRNA expression at indicated time points was detected by qRT-PCR, and IFN-β in supernatants was measured by ELISA. D, murine peritoneal macrophages were transfected with control inhibitor, miR-223 inhibitor, miR-223 inhibitors combined with control siRNA, and miR-223 inhibitors combined with FOXO3 siRNA, respectively, as indicated. After 48 h, macrophages were infected by VSV at m.o.i. 10 for 1 h and washed; intracellular VSV RNA replicates at indicated time points were quantified using qRT-PCR, and the value at 4 h in cells transfected with control inhibitors served as 1-fold control. E, RAW264.7 cells were transfected with FOXO3 overexpression plasmid. After 48 h, FOXO3 mRNA and protein levels were detected by qRT-PCR (left) and immunoblot (right). F, RAW264.7cells were transfected with control (Ctrl) mimics combined with control plasmid, miR-223 mimics combined with control plasmid, or control mimics combined with FOXO3 overexpression plasmid, and miR-223 mimics combined with FOXO3 overexpression plasmid. After 48 h, macrophages were infected by VSV at m.o.i. 10 for 1 h and washed; intracellular VSV RNA replicates at indicated time points were quantified using qRT-PCR, and the level at 4-h time point in cells transfected with control plasmid and control mimics served as 1-fold control. G, proposed positive regulatory loop of type I IFN/miR-223/FOXO3/IFR7 pathway in regulating antiviral innate immunity. Data are the mean ± S.D. (n = 3) of one representative experiment. Similar results were obtained in three independent experiments. ***, p < 0.1; **, p < 0.01; *, p < 0.05; ns, not significant.

Article Snippet: Recombinant mouse IFN-β (rmIFN-β) was from Sino Biological (Beijing, China).

Techniques: Transfection, Infection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Over Expression, Plasmid Preparation, Western Blot

Journal: Nature Communications

Article Title: Functional rare and low frequency variants in BLK and BANK1 contribute to human lupus

doi: 10.1038/s41467-019-10242-9

Figure Lengend Snippet: BLK variants fail to repress type 1 IFN expression. a IFNβ luciferase activity 24 h after co-transfection of HEK293T cells with IRF5, MyD88, and indicated BLK variants. Each data point represents the relative luminometer unit (RLU) relative to a cotransfected control plasmid expressing renilla luciferase, and is the average of three technical replicates. (* p < 0.05, ** p < 0.01, *** p < 0.001 Student t test). b IFNβ luciferase expression in CRISPR-Cas9 edited Ramos cells homozygous for the R131W variant, 24 h after stimulation with resiquimod (R848). Each data point represents the relative luminometer unit (RLU) relative to a cotransfected control plasmid expressing renilla luciferase, and is the average of five technical replicates. (* p < 0.05, ** p < 0.01, Mann–Whitney U ). c IFNβ luciferase activity relative to WT BLK 24 h after co-transfection of HEK293T cells with IRF5, MyD88, and indicated BLK variants found in healthy controls (HC) or SLE patients (SLE). Data points for each variant were normalized to the repression levels of WT BLK. The dotted line indicates 50% of repression by WT BLK (mean and SD). a – c representative of at least three experimental replicates ( p < 0.0001, two-tailed ANOVA). d . Heat map of 34 hierarchically clustered genes within IFN module M1.2. The data are normalized to healthy controls by subtracting out the mean of the healthy controls across all samples. The samples are ordered by condition, illustrating IFN upregulation in both BANK/BLK and non-BLK patients relative to the healthy controls. e For BLK samples, the map shows the percentage of genes within each module (minimum 10%) that are significant (False discovery rate (FDR) ≤ 0.05) and overexpressed (red) or underexpressed (blue) relative to healthy controls. As an example, a module in which 50% of the genes are significantly upregulated and 25% are significantly downregulated would appear on the map as 50–25% = 25% upregulated and would have a light red color

Article Snippet: IFNβ luciferase (Addgene) pRL-CMV (Promega). pCMV-HA-MyD88 was a gift from Bruce Beutler (Addgene plasmid # 12287), pX330-U6-Chimeric_BB-CBh-hSpCas9 was a gift from Feng Zhang (Addgene plasmid # 42230).

Techniques: Expressing, Luciferase, Activity Assay, Cotransfection, Control, Plasmid Preparation, CRISPR, Variant Assay, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Functional rare and low frequency variants in BLK and BANK1 contribute to human lupus

doi: 10.1038/s41467-019-10242-9

Figure Lengend Snippet: Impaired sequestration of TRAF6 by BANK1 increases nuclear IRF5. a Schematic of the role of BANK1 and BLK on regulation of T1 IFN production. Red lines indicate negative regulation. b Indirect immunofluorescence staining of HEK293T cells expressing either wild type (top panels) or W40C (bottom panels) BANK1-V5 cDNA constructs. BANK1 protein was detected using an antibody specific for the V5 tag (red). DNA was stained with DAPI (blue). Scale bar 20 μm. c Quantification of the percentage of HEK293T cells expressing WT or W40C BANK1-V5 with cytoplasmic inclusion bodies. Cells were transfected with 3 μg of the BANK1-V5 cDNA constructs and stained 72 h post transfection. Greyscale inserts show enlargements of the indicated cell sections. Double blind cell counting was carried out on at least 100 cells per experiment with three experimental replicates. d Representative images of indirect immunofluorescence staining of HEK293T cells expressing IRF5, FLAG-TRAF6 and either WT BANK1-V5 (top panel) or W40C BANK1-V5 (bottom panel). IRF5 protein was detected using an antibody specific for IRF5 (green) and BANK1 was detected using an antibody recognizing the V5 tag (red). Greyscale inserts show enlargements of the indicated cell. Scale bar 50 μm. Double blind cell counting was carried out on at least 100 cells per experiment with n = 3 experimental replicates. Bar graph represents quantification of the percentage of nuclear IRF5 as a proportion of the total IRF5 per cell based on indirect immunofluorescence staining of HEK293T cells expressing IRF5, FLAG-TRAF6 and either WT BANK1-V5 or W40C BANK1-V5 (as in ( d )). Quantification was performed using the ImageJ software . (Centre line = median, box = 25th to 75th percentile, whiskers = range; * p < 0.05, ** p < 0.01, Mann–Whitney U , representative of two experimental replicates)

Article Snippet: IFNβ luciferase (Addgene) pRL-CMV (Promega). pCMV-HA-MyD88 was a gift from Bruce Beutler (Addgene plasmid # 12287), pX330-U6-Chimeric_BB-CBh-hSpCas9 was a gift from Feng Zhang (Addgene plasmid # 42230).

Techniques: Immunofluorescence, Staining, Expressing, Construct, Transfection, Cell Counting, Software, MANN-WHITNEY