Cooperative contributions of interferon regulatory factor 1 (IRF1) and IRF8 to interferon-γ-mediated cytotoxic effects on oligodendroglial progenitor cells - PubMed (original) (raw)

Cooperative contributions of interferon regulatory factor 1 (IRF1) and IRF8 to interferon-γ-mediated cytotoxic effects on oligodendroglial progenitor cells

Makoto Horiuchi et al. J Neuroinflammation. 2011.

Abstract

Background: Administration of exogenous interferon-γ (IFNγ) aggravates the symptoms of multiple sclerosis (MS), whereas interferon-β (IFNβ) is used for treatment of MS patients. We previously demonstrated that IFNγ induces apoptosis of oligodendroglial progenitor cells (OPCs), suggesting that IFNγ is more toxic to OPCs than IFNβ. Thus we hypothesized that a difference in expression profiles between IFNγ-inducible and IFNβ-inducible genes in OPCs would predict the genes responsible for IFNγ-mediated cytotoxic effects on OPCs. We have tested this hypothesis particularly focusing on the interferon regulatory factors (IRFs) well-known transcription factors up-regulated by IFNs.

Methods: Highly pure primary rat OPC cultures were treated with IFNγ and IFNβ. Cell death and proliferation were assessed by MTT reduction, caspase-3-like proteinase activity, Annexin-V binding, mitochondrial membrane potential, and BrdU-incorporation. Induction of all nine IRFs was comprehensively compared by quantitative PCR between IFNγ-treated and IFNβ-treated OPCs. IRFs more strongly induced by IFNγ than by IFNβ were selected, and tested for their ability to induce OPC apoptosis by overexpression and by inhibition by dominant-negative proteins or small interference RNA either in the presence or absence of IFNγ.

Results: Unlike IFNγ, IFNβ did not induce apoptosis of OPCs. Among nine IRFs, IRF1 and IRF8 were preferentially up-regulated by IFNγ. In contrast, IRF7 was more robustly induced by IFNβ than by IFNγ. Overexpressed IRF1 elicited apoptosis of OPCs, and a dominant negative IRF1 protein partially protected OPCs from IFNγ-induced apoptosis, indicating a substantial contribution of IRF1 to IFNγ-induced OPC apoptosis. On the other hand, overexpression of IRF8 itself had only marginal proapoptotic effects. However, overexpressed IRF8 enhanced the IFNγ-induced cytotoxicity and the proapoptotic effect of overexpressed IRF1, and down-regulation of IRF8 by siRNA partially but significantly reduced preapoptotic cells after treatment with IFNγ, suggesting that IRF8 cooperatively enhances IFNγ-induced OPC apoptosis.

Conclusions: This study has identified that IRF1 and IRF8 mediate IFNγ-signaling leading to OPC apoptosis. Therapies targeting at these transcription factors and their target genes could reduce IFNγ-induced OPC loss and thereby enhance remyelination in MS patients.

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Figures

Figure 1

Figure 1

IFNβ is far less toxic to OPCs than IFNγ. Phase-contrast images (A-C) and immunocytochemistry for A2B5 (D-F) of rat OPCs treated with GM alone (A, D), GM plus IFNβ (1 kU/ml) (B, E) or GM plus IFNγ (100 ng/ml) (C, F) for 48 h. G, Viability of OPCs cultured with GM (control), GM supplemented with IFNβ (IFNβ), IFNγ (IFNγ), or both (IFNγ + IFNβ) was measured by MTT assay at 48 h after treatment. ** Indicates p < 0.01 compared with control. ** Indicates p < 0.01 compared with control or in comparison between the two groups indicated.

Figure 2

Figure 2

Unlike IFNγ, IFNβ does not induce OPC apoptosis. A-B, Numbers of Annexin-V+/PI- (preapoptotic) cells and Annexin-V+/PI+ (dead) OPCs after treatment with IFNβ or IFNγ for 24h and 48 h. Annexin-V-/PI- (live), preapoptotic, and dead cells were counted by flow cytometry, and shown as percentages of averaged live cell numbers in control at 24 h. C, Caspase-3-like protease activity was measured with the fluorogenic substrate, Ac-DEVD-MCA, in the protein lysates of OPCs treated with GM alone (control, open circles), IFNβ (closed triangles) or IFNγ (closed circles) for 12, 24, and 48 h. ** indicates p < 0.01 compared with control or in comparison between the two groups indicated. D-E, Dose-dependent effects of IFNβ on induction of caspase-3-like proteinase activity and IGTP mRNA in OPCs. OPCs were treated with IFNβ at 0, 0.3, 1, and 3 kU/ml for 24 h, and activity of caspase-3-like proteinase (D) and IGTP mRNA (E) were quantified.

Figure 3

Figure 3

IFNβ inhibits progression of the cell cycle, though to a lesser extent than IFNγ. BrdU-incorporation of OPCs incubated with IFNβ (1 kU/ml, IFNβ) or IFNγ (100 ng/ml, IFNγ) for 24 (open bars) and 48 h (closed bars). OPCs were exposed to a 4 h-BrdU pulse immediately before fixation. ** Indicates p < 0.01 compared with control or in comparison between the two groups indicated.

Figure 4

Figure 4

IFNγ, but not IFNβ, depolarizes OPC mitochondria prior to loss of plasma membrane integrity. Representative histograms of TMRE signals in DAPI-negative OPCs at 24 h after treatment with GM alone (control, A), IFNβ (B) or IFNγ (C). More TMRE-/DAPI- OPCs were detected in the gated area (R3) in the cultures treated with IFNγ for 24 h compared to control and IFNβ-treated cultures. D, TMRE-/DAPI- OPCs became detectable between 12 and 18 h after treatment with IFNγ (closed circle). In contrast, GM alone (control, open circles) or IFNβ (closed triangles) did not increase this population until 24 h after treatment. ** Indicates p < 0.01 compared with control.

Figure 5

Figure 5

IRF1 and IRF8 are preferentially up-regulated in OPCs treated with IFNγ. A-C, Quantitative analysis of induction of IRF1, IRF2, IRF3, IRF6, IRF7, IRF8, and IRF9 mRNA in OPCs before (Cnt 0 h) and at 24 h after incubation with IFNγ (100 ng/ml, G 24 h), IFNβ (1 kU/ml, B 24 h), or medium alone (Cnt 24 h). Each data point was from at least 3 independent experiments. Note that the data are plotted as ratios to copy numbers of GAPDH cDNA on a logarithmic scale. ** Indicates p < 0.01 compared with control at 24 h (Cnt 24 h). D, IRF1 and IRF8 mRNA in OPCs were quantified by real-time PCR at 1, 3, 6, 12, and 24 h after addition of GM alone (control, open circle), IFNγ (100 ng/ml, closed circle) or IFNβ (1 kU/ml, closed triangle). For IRF8, basal IRF8 mRNA levels in the two RNA samples of spleen are shown as positive control (open triangles). At time 0, data from controls are only shown. E, Induction of IRF1 and IRF8 proteins in OPCs was examined at 24 h after treatment with IFNγ and IFNβ by immunoblotting.

Figure 6

Figure 6

Expression constructs used in the study. A, PCMV-IE, immediate-early cytomegalovirus promoter; IRF1 DBD, IRF1 DNA binding domain; IRES, internal ribosome entry site; hrGFP, humanized Renilla GFP; pA, poly adenylation signal sequence. B, Fluorescence signals of hrGFP in the transfected cells became detectable by microscopy as early as 6 h after transfection. C, Fluorescent imaging demonstrates that the IRF1DN-hrGFP fusion protein was localized in the nuclei of transfected OPCs.

Figure 7

Figure 7

Overexpressed IRF1 induces OPC apoptosis, whereas overexpressed IRF8 does not. A, OPCs were transfected with the control vector (PCMV-IE-IRES-hrGFP-pA), the expression construct of rat IRF1 (PCMV-IE-IRF1-IRES-hrGFP-pA), or the expression construct of rat IRF8 (PCMV-IE-IRF8-IRES-hrGFP-pA) by electroporation. These OPCs were incubated with TMRE and DAPI at 6 and 24 h after transfection and analyzed by flow cytometry. B, Representative gating scheme of flow cytometric analysis of preapoptotic (TMRE-/DAPI-) OPCs in the transfected (hrGFP+) and untransfected (hrGFP-) populations at 24 h after transfection with the IRF1 expression construct. Live cells negative for DAPI in the R2 gate were separated into hrGFP + (R3) and hrGFP - (R4) cells. Preapoptotic OPCs were then counted in the R5 and R6 gated areas in hrGFP + (R3) and hrGFP - (R4) populations, respectively. C, Number of preapoptotic (TMRE-/DAPI-) OPCs in transfected (hrGFP+) and untransfected (hrGFP-) populations at 6 and 24 h after transfection with the control vector, IRF1 expression construct or IRF8 expression construct. D, Reduction in live transfected cells (DAPI-/hrGFP+) at 24 h in the cultures transfected with the control vector, IRF1 expression construct or IRF8 expression construct. Due to the different transfection efficiencies among the expression constructs, percentages of live transfected (DAPI-/hrGFP+) cells in total live (DAPI-) cells were calculated in each condition, and are shown as fold changes of the calculated percentages at 6 h after transfection. ** Indicates p < 0.01 (n = 9). E, Overexpressed IRF1 and IRF8 were verified by immunoblotting. GFP-positive cell populations in OPC cultures transfected with the expression constructs of rat IRF1 (IRF1-GFP+) or IRF8 (IRF8-GFP+) were separated from GFP-negative cell populations (IRF1-GFP- and IRF8-GFP-) with a cell sorter, and subjected to immunoblotting for IRF1 and IRF8, respectively. Untransfected OPCs cultured with medium alone (C), and medium containing IFNγ (100 ng/ml, G) were used as negative and positive controls, respectively.

Figure 8

Figure 8

Effects of overexpressed dominant-negative IRF1 and overexpressed IRF8 on IFNγ-induced OPC apoptosis. A, OPCs were transfected by electroporation with the control vector (PCMV-IE-IRES-hrGFP-pA), the expression construct of a dominant-negative form of IRF1 (IRF1DN-hrGFP) which is a fusion protein of IRF1 DNA-binding domain (IRF1DBD) and hrGFP (PCMV-IE-IRF1DBD/hrGFP-pA), and the expression construct of rat IRF8 (PCMV-IE-IRF8-IRES-hrGFP-pA). Cells were cultured with GM for 24 h after transfection, and then treated with GM alone (control) or GM plus IFNγ (100 ng/ml). At 24 h after treatments, cells were stained with TMRE and DAPI, and analyzed by flow cytometry as in Figure. 7. B, Number of preapoptotic (TMRE-/DAPI-) OPCs in the cultures subjected to electroporation with the control vector or the IRF1DN expression construct at 24 h after treatment with IFNγ (100 ng/ml). Transfected (hrGFP+) and untransfected (hrGFP-) populations were analyzed separately, using the same gatings as in Figure. 7. C, Number of live transfected cells (DAPI-/hrGFP+) in the cultures transfected with the control vector, IRF1DN expression construct or IRF8 expression construct after a 24 h IFNγ-treatment (48 h after transfection). Percentages of DAPI-/hrGFP+ cells in total live (DAPI-) cells were calculated in each condition, and are shown as fold changes of the percentages just before addition of IFNγ (24 h after transfection). Note that the same percentages of transfected (hrGFP+) and untransfected (hrGFP-) OPC populations died during 24 h after addition of IFNγ in the control group, whereas less and more transfected OPCs were dead in the IRF1-DN and IRF8 groups, respectively. ** Indicates p < 0.01 in comparison with the corresponding data at 24 h (n = 9).

Figure 9

Figure 9

Selective down-regulation of IRF8 by siRNA protects OPCs from IFNγ-induced OPC apoptosis. A, OPCs were transfected by electroporation with IRF8 siRNA and negative conrol siRNA. Cells were cultured with GM for 3 h after transfection, and then treated with GM alone (control) or GM plus IFNγ (100 ng/ml). At 24 or 48 h after treatments, cells were subjected to western blotting. MTT assay, or stained with TMRE and DAPI followed by the flow cytometric analysis as in Figure. 7. B, Protein levels of IRF8 in the OPCs transfected with negative control siRNA (Neg siRNA) or IRF8 siRNA were examined by immunoblotting at 24 h after addition of IFNγ (+IFNγ, 100 ng/ml). The untransfected OPCs (No siRNA) treated with IFNγ (+IFNγ, 100 ng/ml) or medium alone (C) for 24 h were used as positive and negative control for IRF1 expression, respectively. B, Viability of the OPCs transfected with negative control siRNA (control siRNA, open bar) or IRF8 siRNA (closed bar) was measured by MTT assay at 48 h after treatment with GM alone (control) or GM plus IFNγ (100 ng/ml). C, Number of preapoptotic (TMRE-/DAPI-) OPCs in the cultures subjected to electroporation with negative control siRNA (control siRNA, open bar) or IRF8 siRNA (closed bar) at 24 h after treatment with IFNγ (100 ng/ml). ** Indicates p < 0.01 (n = 9).

Figure 10

Figure 10

IRF8 enhances the proapoptotic effect of IRF1 in the absence of IFNγ. A, OPCs were co-transfected with the following combinations of expression constructs by electroporation; The control GFP vector (PCMV-IE-IRES-hrGFP-pA) plus the rat IRF8 expression construct without GFP reporter (PCMV-IE-IRF8-pA) (GFP+IRF8), the rat IRF1 expression construct with GFP reporter (PCMV-IE-IRF1-IRES-hrGFP-pA) plus the rat IRF8 expression construct without GFP reporter (PCMV-IE-IRF8-pA) (IRF1+IRF8), or PCMV-IE-IRF1-IRES-hrGFP-pA plus the empty vector (PCMV-IE-pA) (IRF1+empty). These OPCs were incubated with TMRE and DAPI at 6 and 24 h after transfection and analyzed by flow cytometry. B, Number of preapoptotic (TMRE-/DAPI-) OPCs at 24 h after electroporation with GFP+IRF8, IRF1+empty, and IRF1+IRF8. Transfected (hrGFP+) and untransfected (hrGFP-) populations were analyzed separately, using the same gatings as in Figure. 7. ** Indicates p < 0.01 compared with GFP- (non-transfected) counterparts, or in comparison between the two groups indicated. **C**, Reduction in live transfected cells (DAPI-/hrGFP+) at 24 h in the cultures transfected with GFP+IRF8, IRF1+empty, or IRF1+IRF8. Percentages of DAPI-/hrGFP+ cells in total live (DAPI-) cells were calculated in each condition, and are shown as fold changes of those at 6 h after transfection. N.S. indicates p > = 0.05 between the two groups indicated.

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References

    1. Watanabe M, Hadzic T, Nishiyama A. Transient upregulation of Nkx2.2 expression in oligodendrocyte lineage cells during remyelination. Glia. 2004;46:311–322. doi: 10.1002/glia.20006. - DOI - PubMed
    1. Islam MS, Tatsumi K, Okuda H, Shiosaka S, Wanaka A. Olig2-expressing progenitor cells preferentially differentiate into oligodendrocytes in cuprizone-induced demyelinated lesions. Neurochem Int. 2009;54:192–198. doi: 10.1016/j.neuint.2008.10.011. - DOI - PubMed
    1. Reynolds R, Dawson M, Papadopoulos D, Polito A, Di Bello IC, Pham-Dinh D, Levine J. The response of NG2-expressing oligodendrocyte progenitors to demyelination in MOG-EAE and MS. J Neurocytol. 2002;31:523–536. doi: 10.1023/A:1025747832215. - DOI - PubMed
    1. Agresti C, D'Urso D, Levi G. Reversible inhibitory effects of interferon-gamma and tumour necrosis factor-alpha on oligodendroglial lineage cell proliferation and differentiation in vitro. Eur J Neurosci. 1996;8:1106–1116. doi: 10.1111/j.1460-9568.1996.tb01278.x. - DOI - PubMed
    1. Andrews T, Zhang P, Bhat NR. TNFalpha potentiates IFNgamma-induced cell death in oligodendrocyte progenitors. J Neurosci Res. 1998;54:574–583. doi: 10.1002/(SICI)1097-4547(19981201)54:5<574::AID-JNR2>3.0.CO;2-0. - DOI - PubMed

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