Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Oct:13:633-645.
doi: 10.1016/j.redox.2017.07.016. Epub 2017 Jul 29.

Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells

Zsofia Agod  1 , Tünde Fekete  1 , Marietta M Budai  1 , Aliz Varga  1 , Attila Szabo  2 , Hyelim Moon  1 , Istvan Boldogh  3 , Tamas Biro  1 , Arpad Lanyi  2 , Attila Bacsi  2 , Kitti Pazmandi  4
Affiliations

Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells

Zsofia Agod et al. Redox Biol. 2017 Oct.

Abstract

Mitochondrial reactive oxygen species (mtROS) generated continuously under physiological conditions have recently emerged as critical players in the regulation of immune signaling pathways. In this study we have investigated the regulation of antiviral signaling by increased mtROS production in plasmacytoid dendritic cells (pDCs), which, as major producers of type I interferons (IFN), are the key coordinators of antiviral immunity. The early phase of type I IFN production in pDCs is mediated by endosomal Toll-like receptors (TLRs), whereas the late phase of IFN response can also be triggered by cytosolic retinoic acid-inducible gene-I (RIG-I), expression of which is induced upon TLR stimulation. Therefore, pDCs provide an ideal model to study the impact of elevated mtROS on the antiviral signaling pathways initiated by receptors with distinct subcellular localization. We found that elevated level of mtROS alone did not change the phenotype and the baseline cytokine profile of resting pDCs. Nevertheless increased mtROS levels in pDCs lowered the TLR9-induced secretion of pro-inflammatory mediators slightly, whereas reduced type I IFN production markedly via blocking phosphorylation of interferon regulatory factor 7 (IRF7), the key transcription factor of the TLR9 signaling pathway. The TLR9-induced expression of RIG-I in pDCs was also negatively regulated by enhanced mtROS production. On the contrary, elevated mtROS significantly augmented the RIG-I-stimulated expression of type I IFNs, as well as the expression of mitochondrial antiviral-signaling (MAVS) protein and the phosphorylation of Akt and IRF3 that are essential components of RIG-I signaling. Collectively, our data suggest that increased mtROS exert diverse immunoregulatory functions in pDCs both in the early and late phase of type I IFN responses depending on which type of viral sensing pathway is stimulated.

Keywords: Antiviral response; Endosomal TLR signaling; Mitochondrial ROS; Plasmacytoid dendritic cell; RIG-I signaling; Type I interferon.

PubMed Disclaimer

Figures

fx1
Graphical abstract
Fig. 1
Fig. 1
Generation of elevated level of mtROS in GEN2.2 cells without affecting the cell viability. Cells were loaded with MitoSox™ Red mitochondrial superoxide indicator and then treated with AMA (0.5 μg/ml) for 6 h to increase the production of mtROS. As a control AMA was used in combination with MitoTEMPO (300 μM) that limits mtROS accumulation. MitoTEMPO was added 1 h prior to and along with the AMA treatments. The fluorescence intensity of MitoSox™ Red dye correlates with the level of mtROS generated in the cells. The changes in fluorescence intensities of MitoSox™ Red dye were monitored by flow cytometry. A representative histogram (A) and the means ± SD (B) of eight independent experiments are shown. The percentage of dead cells was determined by 7-AAD staining. (C) Dot plots are representatives of eight independent experiments. ****p < 0.0001 vs. control, ####p < 0.0001 vs. AMA. AMA: Antimycin-A.
Fig. 2
Fig. 2
Phenotypical analysis of AMA-exposed GEN2.2 cell. Cells were treated with AMA (0.5 μg/ml) and CpG-A (1 μM) separately and in combination or were left untreated. Following 6 h of stimulation the expression levels of CD40 (A), CD80 (B), CD86 (C) and HLA-DQ (D) cell surface proteins were analyzed by flow cytometry. Relative fluorescence intensity values were calculated using the respective isotype-matched control antibodies. The bars represent fold changes compared to the untreated control and data are expressed as the mean ± SD of four independent experiments. *p < 0.05 vs. control. AMA: Antimycin-A.
Fig. 3
Fig. 3
Pro-inflammatory cytokine and chemokine secretion of GEN2.2 cells exposed to AMA. Cells were stimulated with AMA (0.5 μg/ml) and CpG-A (1 μM) separately and in combination. After 6 h cell culture supernatants were collected and the levels of IL-6 (A), TNF-alpha (B) and IL-8 (C) proteins were assessed by ELISA. Data are presented as means ± SD of four independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001 vs. control, #p < 0.05, ###p < 0.001 vs. CpG-A. AMA: Antimycin-A.
Fig. 4
Fig. 4
AMA effects on the production of type I IFNs and phosphorylation of IRF7 in TLR9-activated GEN2.2 cells. Cells were stimulated with the TLR9 agonist CpG-A (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A). The level of secreted IFN-α cytokine was determined by ELISA (B). Cells were stimulated for 30 min as described above then the levels of the phosphorylated (p-IRF7) and native form of IRF7 were determined by Western blotting (C, D). Bars represent the means ± SD of three individual experiments (A, B, D) and a representative blot is shown (C). ***p < 0.001, ****p < 0.0001 vs. control, #p < 0.05, ##p < 0.01, ####p < 0.0001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.
Fig. 5
Fig. 5
Dose- and time-dependent induction of cytosolic RIG-I receptor in GEN2.2 cells and the expression of RIG-I in AMA-exposed cells. In order to induce the cytosolic expression of RIG-I GEN2.2 cells were treated with increasing concentration of the specific TLR9 ligand, CpG-A (ranging from 0.01 to 0.5 μM). After 16 h the presence of RIG-I was detected in the cell lysates by Western blotting (A, B). To evaluate the time-dependent induction of RIG-I, GEN2.2 cells were exposed to 0.25 μM of CpG-A then the expression of RIG-I was measured in different time points (C, D). Finally cells were exposed to 0.25 μM of CpG-A in the presence or absence of AMA (0.5 μg/ml) or left untreated, for 16 h. Then cells were lysed and the protein level of RIG-I was assessed (E, F). Representative blots (A, C, E) and the means ± SD of four independent experiments (B, D, F) are shown. ***p < 0.001, ****p < 0.0001 vs. controls, ###p < 0.001 vs. CpG-A. AMA: Antimycin-A, ND: not determined.
Fig. 6
Fig. 6
RIG-I and TLR9 induced type I IFN production in CpG-A-pre-conditioned GEN2.2 cells in the presence of AMA. Cells were pre-treated with CpG-A (0.25 μM) for 16 h to induce the cytosolic expression of RIG-I. Following thorough washing steps cells were re-exposed to the specific RIG-I ligand 5′ppp-dsRNA (RIGL, 1 μg/ml) in the presence or absence of AMA (0.5 μg/ml). The IFNA1 mRNA expression level was determined by real-time PCR after 3 h (A) and IFN-α protein level was assessed by ELISA after 6 h (B). In parallel experiments the re-activation of the cells was carried out with high dose of CpG-A (1 μM) in combination or not with AMA (0.5 μg/ml). IFNA1 mRNA (C) and IFN-α protein levels (D) were measured as in (A) and (B), respectively. Data are presented as means ± SD of four individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control, #p < 0.05 vs. RIGL and ##p < 0.01 vs. CpG-A. AMA: Antimycin-A, ND: not determined.
Fig. 7
Fig. 7
Effects of AMA treatments on the first and second waves of type I IFN production in primary human pDCs. Freshly isolated primary pDCs were stimulated with the TLR9 agonist CpG-A (1 μM) in the presence or absence of AMA (0.5 μg/ml). After 6 h of stimulation the mRNA expression of IFNA1 was measured by real-time PCR (A) and the level of secreted IFN-α cytokine was determined by ELISA (B). To investigate the effects of AMA on the second wave of type I IFN responses, primary pDCs were pre-treated with CpG-A for 16 h then after thorough washing steps cells were re-exposed to 5′ppp-dsRNA (RIGL, 1 μg/ml) in the presence or absence of AMA (0.5 μg/ml). After 3 h the IFNA1 mRNA expression level was determined by real-time PCR (C) and after 6 h IFN-α protein level was assessed by ELISA (D). Data are presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control, #p < 0.05 vs. CpG-A or RIGL and ##p < 0.01 vs. CpG-A or RIGL. AMA: Antimycin-A, ND: not determined.
Fig. 8
Fig. 8
Analysis of signaling events in RIG-I-activated GEN2.2 cells in the presence or absence of AMA. Cytosolic RIG-I expression was induced in GEN2.2 by pre-treatment with CpG-A (0.25 μM) for 16 h then cells were stimulated with the specific RIG-I ligand 5′ppp-dsRNA (RIGL, 1 μg/ml) in the presence or absence of AMA (0.5 μg/ml). After 2 h of stimulation the expression of the MAVS adaptor protein (A, B) and after 30 min of stimulation the phosphorylation of IRF3 (C, D) and Akt (E, F) were determined by Western blotting. Representative blots (A, C, E) and the means ± SD of four independent experiments (B, D, F) are shown. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control, #p < 0.05, ##p < 0.01 vs. RIGL. AMA: Antimycin-A.
Fig. 9
Fig. 9
Overview of the effects of elevated level of mtROS on the endosomal TLR-, and cytosolic RIG-I-mediated pDC activation. Upon infection cells are affected by many various exogenous and endogenous stress factors leading to higher respiratory activity of the mitochondria and subsequent increase in mtROS production in the cells. We found that the elevated level of mtROS has a negative impact on the first wave of type I IFN responses mediated by endosomal TLRs whereas it has a positive effect on the second wave of type I IFN production guided by cytosolic RIG-I receptor in pDCs. Thus, our data indicate opposing regulatory role of mtROS depending on the receptor-context in pDCs.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Schieber M., Chandel N.S. ROS function in redox signaling and oxidative stress. Curr. Biol. 2014;24(10):R453–R462. - PMC - PubMed
    1. Turrens J.F. Mitochondrial formation of reactive oxygen species. J. Physiol. 2003;552(Pt 2):335–344. - PMC - PubMed
    1. Shadel G.S., Horvath T.L. Mitochondrial ROS signaling in organismal homeostasis. Cell. 2015;163(3):560–569. - PMC - PubMed
    1. Li X., Fang P., Mai J., Choi E.T., Wang H., Yang X.F. Targeting mitochondrial reactive oxygen species as novel therapy for inflammatory diseases and cancers. J. Hematol. Oncol. 2013;6:19. - PMC - PubMed
    1. Muller F.L., Liu Y., Van Remmen H. Complex III releases superoxide to both sides of the inner mitochondrial membrane. J. Biol. Chem. 2004;279(47):49064–49073. - PubMed

LinkOut - more resources