Figure 3.
Figure 3. Expression and function of myeloid-specific lentiviral Ifnγr1 overexpression in Ifnγr1−/− macrophages. (A) Schematic representation of the lentiviral vectors expressing Ifnγr1 cDNA by a synthetic MSP (Lv.MSP.Ifnγr1) or an miRNA223 promoter (Lv.miR223.Ifnγr1), both coupled to a GFP reporter via an IRES. (B) Histogram depicting IFNγR1 expression in Ifnγr1−/−, WT, Lv.MSP.Ifnγr1-transduced, and Lv.miR223.Ifnγr1-transduced macrophages. Bar graphs depict the mean fluorescence intensity (MFI) of IFNγR1 (CD119; n = 3; one-way ANOVA using Dunnett’s multiple comparisons post hoc testing). (C) Representative brightfield, GFP–, and autofluorescence photos of mock-transduced, Lv.miR223.Ifnγr1-transduced, and Lv.MSP.Ifnγr1-transduced macrophages. Scale bars represent 50 μm. (D) Representative histograms depicting MHC-II upregulation on IFN-γ–stimulated vs unstimulated WT, Ifnγr1−/−, Lv.MSP.Ifnγr1-transduced, and Lv.miR223.Ifnγr1-transduced macrophages. Bar graphs depict summarized results of 4 independent experiments (one-way ANOVA using Dunnett’s multiple comparisons post hoc testing). (E) Representative histograms depicting CD86 upregulation on IFN-γ–stimulated vs unstimulated WT, Ifnγr1−/−, Lv.MSP.Ifnγr1-transduced, and Lv.miR223.Ifnγr1-transduced macrophages. Bar graphs depict summarized results of 4 independent experiments (one-way ANOVA using Dunnett’s multiple comparisons post hoc testing). (F) Relative mRNA expression of Irf1 and Nos2 in IFN-γ–stimulated WT, Ifnγr1−/−, Lv.MSP.Ifnγr1-transduced, and Lv.miR223.Ifnγr1-transduced macrophages normalized to unstimulated samples (n = 4). (G) IFN-γ internalization of WT, Ifnγr1−/−, Lv.MSP.Ifnγr1-transduced, and Lv.miR223.Ifnγr1-transduced macrophages over 32 hours (normalized to 0 hours; n = 3; two-way ANOVA using Tukey’s multiple comparisons post hoc testing). (H) Bacterial burden of BCG in Lv.MSP.Ifnγr1-transduced and Lv.miR223.Ifnγr1-transduced macrophages 24 hours after infection sorted into positive and negative groups for GFP expression (normalized to uptake value 0 hours after infection; n = 3). *P ≤ .05; **P ≤ .01; ****P ≤ .0001. For all bars, mean and SEM are shown.

Expression and function of myeloid-specific lentiviral Ifnγr1 overexpression in Ifnγr1−/−macrophages. (A) Schematic representation of the lentiviral vectors expressing Ifnγr1 cDNA by a synthetic MSP (Lv.MSP.Ifnγr1) or an miRNA223 promoter (Lv.miR223.Ifnγr1), both coupled to a GFP reporter via an IRES. (B) Histogram depicting IFNγR1 expression in Ifnγr1−/−, WT, Lv.MSP.Ifnγr1-transduced, and Lv.miR223.Ifnγr1-transduced macrophages. Bar graphs depict the mean fluorescence intensity (MFI) of IFNγR1 (CD119; n = 3; one-way ANOVA using Dunnett’s multiple comparisons post hoc testing). (C) Representative brightfield, GFP–, and autofluorescence photos of mock-transduced, Lv.miR223.Ifnγr1-transduced, and Lv.MSP.Ifnγr1-transduced macrophages. Scale bars represent 50 μm. (D) Representative histograms depicting MHC-II upregulation on IFN-γ–stimulated vs unstimulated WT, Ifnγr1−/−, Lv.MSP.Ifnγr1-transduced, and Lv.miR223.Ifnγr1-transduced macrophages. Bar graphs depict summarized results of 4 independent experiments (one-way ANOVA using Dunnett’s multiple comparisons post hoc testing). (E) Representative histograms depicting CD86 upregulation on IFN-γ–stimulated vs unstimulated WT, Ifnγr1−/−, Lv.MSP.Ifnγr1-transduced, and Lv.miR223.Ifnγr1-transduced macrophages. Bar graphs depict summarized results of 4 independent experiments (one-way ANOVA using Dunnett’s multiple comparisons post hoc testing). (F) Relative mRNA expression of Irf1 and Nos2 in IFN-γ–stimulated WT, Ifnγr1−/−, Lv.MSP.Ifnγr1-transduced, and Lv.miR223.Ifnγr1-transduced macrophages normalized to unstimulated samples (n = 4). (G) IFN-γ internalization of WT, Ifnγr1−/−, Lv.MSP.Ifnγr1-transduced, and Lv.miR223.Ifnγr1-transduced macrophages over 32 hours (normalized to 0 hours; n = 3; two-way ANOVA using Tukey’s multiple comparisons post hoc testing). (H) Bacterial burden of BCG in Lv.MSP.Ifnγr1-transduced and Lv.miR223.Ifnγr1-transduced macrophages 24 hours after infection sorted into positive and negative groups for GFP expression (normalized to uptake value 0 hours after infection; n = 3). *P ≤ .05; **P ≤ .01; ****P ≤ .0001. For all bars, mean and SEM are shown.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal