Fig 1: Type I IFN Signaling Affects Mitochondrial Metabolism Leading to Proinflammatory Oxidative Stress and Ionic Alterations of Lysosomal Compartments(Α) Scatterplot (R/Bioconductor) of log2-transformed RNA-seq expression values of all human genes between active SLE (n = 7) and healthy control (n = 5) CD14+ monocytes. All non-differentially expressed genes are depicted as gray colored. Selected genes related to type I IFN signature, oxidative stress, metabolism, antigen presentation, and differentiation are noted in the up (red color) and down (blue color) differentially expressed genes.(B) Relative mitochondrial O2− levels of healthy CD14+ monocytes ± IFNα, rapam. were assessed by flow cytometry using mitoSOX dye (n = 5) One representative histogram overlay is shown and gates indicating low and high mtROS levels are depicted. Averages of % of cells in the mitoSOX high population are graphed. Datasets were analyzed using paired Student’s t test.(C) Concentration of intracellular ATP concentration ± IFNα, rapam., or mitoTEMPO in lysates of CD14+ monocytes (n = 5). Datasets were analyzed using paired Student’s t test.(D) Confocal microscopy for Lysotracker DND99 in CD14+ monocytes from healthy donors ± IFNα, mitoTEMPO. One representative result is depicted. Averages of MFI per cell are graphed (n = 3). Datasets were analyzed using paired Student’s t test.(E) Levels of HLA-DR and CD86 expression measured by flow cytometry in CD14+ monocytes upon IFNα signaling and mtROS scavenging. GeoMFI averages are plotted (n = 3). Datasets were analyzed using paired Student’s t test.(F) Concentrations of secreted IL6 and TNFα measured by ELISA in culture supernatants of CD14+ monocytes (n = 7) cultured as in (D) are graphed. Scale bar, 5 μM. Results are expressed as mean +SEM. ∗p < 0.05, ∗∗p < 0.005.
Fig 2: PS ODNs inhibit TLR3 and TLR7. PBMCs from healthy donors (n=6–7) were treated with 1.4 μm IRS661, 2.8 μm IRS869, 2.8 μm cODN1, 2.8 μm cODN2 or untreated and then stimulated with 25 μg ml−1 Poly (I:C) (TLR3; a, b) or 0.3 μg ml−1 gardiquimod (TLR7; c, d) or left unstimulated for 24 h at 37 °C. Solid lines represent medians and dotted lines represent limit of detection. *P<0.05, **P<0.01, ***P<0.001 compared with TLR ligand-only treated condition. cODN, control ODN; IFN, interferon; IP-10, interferon gamma-induced protein 10; ODN, oligonucleotide.
Fig 3: IFNα-Mediated Lysosomal Dysfunction Impedes Mitochondrial Clearance and Leads to mtDNA Accumulation(Α) Confocal microscopy for Mitotracker CMXRos dye staining in freshly isolated CD14+ monocytes from healthy donors (n = 8) and SLE patients (n = 8). One representative result is depicted. Averages of MFI per cell are graphed. Datasets were analyzed using non-parametric Mann-Whitney U test.(Β) TaqMan qPCR analysis for mtDNA content in freshly isolated monocytes of healthy donors (n = 5) and SLE patients (n = 5), expressed as a ratio of the Cq values of genomic DNA (gDNA) (β2 m)/mtDNA (mt minArc). Datasets were analyzed using non-parametric Mann-Whitney U test.(C) Relative mRNA expression of PINK1 compared to GAPDH in CD14+ monocytes from healthy donors (n = 8) and SLE patients (n = 9). Datasets were analyzed using non-parametric Mann-Whitney U test.(D) Confocal microscopy for JC-1 dye-staining in CD14+ monocytes from healthy donors ± IFNα (n = 4). One representative result is depicted. Average of MFI of J-aggregates per cell are shown. Datasets were analyzed using paired Student’s t test.(E) Confocal Microscopy for Mitotracker CMXRos dye staining in freshly isolated CD14+ monocytes from healthy donors (n = 20) +/− IFN-α for 18h. One representative result is depicted. Αverages of MFI per cell are graphed.(F) Relative mRNA expression of PINK1 compared to GAPDH in CD14+ monocytes from healthy donors (n = 10).(G) Taqman QPCR analysis for mtDNA content in freshly isolated monocytes of healthy donors (n = 5) +/− IFN-α, rapam for 18 h expressed as a ratio of the Cq values of gDNA (β2m)/ mt DNA (mt minArc).Scale bar, 5 μM. Results are expressed as mean + SEM. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, ∗∗∗∗p < 0.00005. Datasets were analyzed using paired Student’s t test. As in (A) for CD14+ monocytes from healthy donors treated with IFN-α +/− rapam.
Fig 4: Type I IFN Signaling Impairs Autophagolysosomal Degradation in Healthy CD14+ MonocytesCD14+ monocytes from healthy donors were treated with recombinant IFNα (400 ng/mL) +/− CQ (46.1 μΜ), rapamycin (rapam.; 1 μΜ) as depicted.(A) Relative mRNA expression of ATG5 compared to GAPDH (n = 9 healthy donors). Datasets were analyzed using non-parametric Mann-Whitney U test.(B) Western blot analysis for LC3B lipidation and SQSTM1/P62 protein levels in whole cell lysates (n = 10 healthy donors). Relative intensities of LC3II to LC3I and SQSTM1/P62 to actin are shown. Datasets were analyzed using non-parametric Mann-Whitney U test.(C) Relative quantity of SQSTM1/P62mRNA levels as in (A). Datasets were analyzed using non-parametric Mann-Whitney U test.(D) Representative western blot analysis of autophagic flux upon IFNα signaling.(E) Confocal microscopy for LC3B555, SQSTM1/P62488, LAMP-1633, and DAPI in CD14+ monocytes from healthy donors treated as indicated. One representative result is depicted. % co-localization of LC3B and P62 puncta is shown for monocytes cultured for 18 hr (n = 8).(F) Confocal microscopy for Lysotracker DND99TM ± rapam. One representative result is depicted. Mean fluorescence intensity ratios are shown (n = 6). Datasets were analyzed using paired Student’s t test.(G) Relative cathepsin D activity ± rapam. (n = 5). Datasets were analyzed using paired Student’s t test.(H) Analysis of autophagic flux assessment with western blotting. Fold change of P62/actin intensity ratios are graphed (n = 4). Scale bar, 5 μM. Results are expressed as mean +SEM. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005. Datasets were analyzed using paired Student’s t test.
Fig 5: The ELISA for IFN-α,-β and -γ are shown in order in three graphs (a), (b) & (c). TLR-3 down regulated cells showed no increase in release of the soluble cytokines upon reovirus infection at a dose of 5 MOI. On the contrary, HCT116 control cells did show a rise in release of the three classes of interferons upon reovirus infection at 5 MOI. In case of IFN-α there was a significant increase (p≤ 0.05) of cytokine productions at all 4 time points namely 6, 12, 24 and 48 hours as represented by *. In case of IFN-β the significance (p≤ 0.05) was observed in control cells treated and untreated at 6 and 12 hours where as the IFN-γ showed a significant difference in the same group at 24 and 48 hours.
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