Research ArticleImmunology

Aging impairs both primary and secondary RIG-I signaling for interferon induction in human monocytes

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Science Signaling  12 Dec 2017:
Vol. 10, Issue 509, eaan2392
DOI: 10.1126/scisignal.aan2392
  • Fig. 1 Monocytes from older humans exhibit cell-intrinsic impairment of RIG-I–induced type I IFN expression.

    Human monocytes enriched from the blood of younger (age, 20 to 30 years; n = 18) and older (age, 65 to 89 years; n = 12) healthy donors were transfected with a retinoic acid–inducible gene I (RIG-I)–specific 5′-triphosphate (5′-ppp) 14–base pair (bp) double-stranded RNA (dsRNA) ligand. (A) RNA was isolated from monocytes 12 hours after stimulation and analyzed by quantitative polymerase chain reaction (qPCR) to measure IFNB and IFNL1 expression. Expression values for each donor were normalized to HPRT. (B) Monocytes were treated with Protein Transport Inhibitor Cocktail 3 hours after stimulation, and at 6 hours after stimulation, cells were fixed, permeabilized, and labeled for intracellular interferon-β (IFN-β) or (C) tumor necrosis factor–α (TNF-α) protein, the median fluorescence intensity (MFI) of which was measured using flow cytometry. (D) Representative flow cytometry plots of fixed and permeabilized monocytes stained for both intracellular TNF-α and IFN-α. (E and F) Using these plots, the frequency of TNF-α+ monocytes (E) and IFN-α+ monocytes (F) was calculated. Data are means ± SEM. **P < 0.01, Student’s t test.

  • Fig. 2 TBK1, IRF3, and IRF7 phosphorylation in response to RIG-I stimulation is impaired in older monocytes.

    (A and B) Monocytes from younger (n = 6) and older (n = 5) donors were transfected with a RIG-I–specific ligand and labeled for intracellular phosphorylated IFN regulatory transcription factor 3 (pIRF3) (A) or phosphorylated IRF7 (pIRF7) (B) at the indicated time points (younger, n = 9; older, n = 7). (C) Phosphorylated TBK1 (pTBK1) levels in monocytes from older (n = 6) or younger (n = 6) donors was assessed 30 min after transfection with a RIG-I–specific ligand. Data are means ± SEM. *P < 0.05, Student’s t test, two-way analysis of variance (ANOVA), or, for pIRF7, a linear mixed model (with a Toeplitz covariance structure).

  • Fig. 3 Increased proteasomal degradation of TRAF3 in older monocytes contributes to impaired IFN induction.

    (A) Tumor necrosis factor receptor–associated factor 3 (TRAF3) and β-actin abundance in older (n = 6) and younger (n = 6) unstimulated monocytes was measured by Western blotting, and (B) the abundance of TRAF3 was quantified by densitometry and normalized to β-actin using ImageJ. (C) TRAF3 expression was measured by qPCR in unstimulated younger (n = 19) and older (n = 11) monocytes. Expression values for each donor were normalized to HPRT. (D and E) Older monocytes (n = 12) were treated with bortezomib for 4 hours before TRAF3 and β-actin abundance was measured by Western blotting (D) and quantified by densitometry (E). (F) Older monocytes (n = 12) were pretreated with bortezomib for 4 hours before transfection with a RIG-I–specific ligand for 6 hours, and IFNB expression was quantified by qPCR. (G) TRAF3 was immunoprecipitated from lysates of old (n = 5) and young (n = 7) human monocytes, and both input and TRAF3 immunoprecipitates (IP: TRAF3) were subjected to Western blotting using antibodies recognizing TRAF3 (IB: TRAF3) and K48-polyubiquitin (IB: K48-Ub). (H) Quantification of K48-polyubiquitin by densitometry. Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, paired or unpaired Student’s t test.

  • Fig. 4 RNA-seq reveals impairment of the amplification phase of the IFN response to RIG-I stimulation in older monocytes.

    Human monocytes from younger (n = 2) and older (n = 3) healthy donor blood were transfected with a RIG-I–specific 5′-ppp 14-bp dsRNA ligand. After 6 hours, RNA was isolated from these cells and was used for exploratory RNA sequencing (RNA-seq). (A) Volcano plot of differentially regulated genes. (B and C) Normalized values of transcripts encoding both types I and III IFN (B) and IRFs (C) were compiled into heat maps using Microsoft Excel to highlight trends in the differential induction of these genes.

  • Fig. 5 Impaired IRF8 induction compromises amplification of the IFN response in older monocytes, and restoring IRF normalizes this response.

    (A and B) Monocytes from younger (n = 19) and older (n = 12) human donors were transfected with a RIG-I–specific ligand (A) or infected with PR8 IAV (B), and IRF8 expression was measured by qPCR at the indicated time points. Expression values for each donor were normalized to HPRT. (C and D) Younger (n = 4) and older (n = 3) monocytes were transfected with a RIG-I–specific ligand for 6 hours, IRF8 protein abundance was measured by flow cytometry (C), and mean fluorescence intensity (MFI) values were quantified (D). Data are representative of two independent experiments. (E) Monocytes from five younger donors were left untreated or were treated with cycloheximide (CHX) for 4 hours before mock transfection or RIG-I stimulation for 6 hours, after which IRF8 expression was quantified by qPCR. NS, not significant. (F and G) Monocytes from younger donors were treated with an IRF8-specific small interfering RNA (siRNA) or RNA-induced silencing complex (RISC)–free control for 48 hours and then stimulated with a RIG-I–specific ligand. IRF8 expression was quantified by qPCR after 6 hours of RIG-I stimulation (F), and IFN-β secreted into the supernatant was quantified by enzyme-linked immunosorbent assay (ELISA) after 12 hours of RIG-I stimulation (G). Data are representative of two independent experiments. (H and I) Monocytes from older donors (n = 16) were transfected with an IRF8 or control lentiviral (LV) construct for 48 hours and then stimulated with a RIG-I–specific ligand. IRF8 expression was quantified by qPCR after 6 hours of RIG-I stimulation (H), and secreted IFN-β was measured by ELISA after 12 hours of RIG-I stimulation (I). (J) Monocytes from younger (n = 7) and older (n = 7) donors were transfected with a control or IRF8 LV construct for 48 hours and then stimulated with a RIG-I–specific ligand for 6 hours, and IRF7 MFI was quantified by flow cytometry. Hp14ppp, 14-bp hairpin dsRNA bearing a 5′-ppp motif. Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, unpaired or paired Student’s t test or two-way repeated-measures ANOVA.

  • Fig. 6 A model of age-related impairment of IFN Induction in monocytes.

    During the primary phase of the IFN response (minutes to hours after IAV infection or RIG-I stimulation), RIG-I mediates phosphorylation of the transcription factor IRF3 through a mechanism that depends on TRAF3. Once phosphorylated, IRF3 homodimerizes and mediates the rapid transcription of genes encoding type I IFNs, which are secreted from the cell, and promotes the expression of other primary transcripts such as IRF8. Aging is associated with decreased TRAF3 protein abundance as a consequence of increased K48-polyubiquitin–mediated TRAF3 proteasomal degradation, which impairs IRF3 phosphorylation and consequent IFN secretion. During the secondary feedback phases of the IFN response, secreted IFNs and continued RIG-I signaling promote the further induction of IRF8 within the cell. IFNs produced during the primary response activate the IFN-α/β receptor (IFNAR) to stimulate the assembly of the IFN-stimulated gene factor 3 (ISGF3) complex, which further stimulates the production of IRF8. IRF8 cooperates with the other IRF family members to amplify the production of IFNs, producing a robust antiviral IFN response. Aging impairs IRF8 induction, thereby further compromising IFN production.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/509/eaan2392/DC1

    Fig. S1. Monocyte purity and plasmacytoid DC numbers are comparable between age groups.

    Fig. S2. IFN Induction in response to dsRNA transfection depends on a 5′-ppp motif in human monocytes.

    Fig. S3. Basal IFNB expression is comparable in human monocytes from older and younger donors.

    Fig. S4. IFN-β pretreatment is insufficient to ablate age-related differences in IFN-β secretion upon RIG-I stimulation.

    Fig. S5. Basal levels of RIG-I, IRF3, and IRF7 protein are comparable in older and younger monocytes.

    Fig. S6. IRF3 phosphorylation is impaired in monocytes from older donors.

    Fig. S7. Basal TRAF3 protein levels are positively correlated with IFN-β secretion in response to RIG-I stimulation.

    Fig. S8. Bortezomib improves antiviral responses in monocytes from older donors.

    Fig. S9. Lack of age-related differences in IFN-β secretion in response to the cGAMP stimulation of human monocytes.

    Fig. S10. RNA-seq highlights the differential regulation of RIG-I, IRF, and IAV-related signaling pathways in older RIG-I–stimulated monocytes.

    Fig. S11. Amplification-stage increase in IRF7 protein is defective in older RIG-I–stimulated monocytes.

    Fig. S12. Basal IRF8 protein and RNA levels are comparable in old and young monocytes.

    Fig. S13. Inducible IRF8 expression is age-dependent.

    Fig. S14. Inducible IRF8 levels are positively correlated with RIG-I–inducible IFN-β secretion.

    Fig. S15. Bortezomib treatment of older monocytes enhances IRF8 induction upon RIG-I stimulation.

    Fig. S16. IFN-β–stimulated gene induction and inducible protection from influenza is preserved with age in human monocytes.

    Fig. S17. IRF8 is an IFN-inducible gene, and its induction is impaired in older monocytes.

    Fig. S18. Lack of age-related difference in CpG DNA methylation of the IRF8 gene in human monocytes.

    Table S1. Human donor characteristics.

    Table S2. Differences in IFN expression in the RNA-seq data from RIG-I–stimulated older and younger monocytes.

    Table S3. Differences in IRF expression in the RNA-seq data from RIG-I–stimulated older and younger monocytes.

  • Supplementary Materials for:

    Aging impairs both primary and secondary RIG-I signaling for interferon induction in human monocytes

    Ryan D. Molony, Jenny T. Nguyen, Yong Kong, Ruth R. Montgomery, Albert C. Shaw, Akiko Iwasaki*

    *Corresponding author. Email: akiko.iwasaki{at}yale.edu

    This PDF file includes:

    • Fig. S1. Monocyte purity and plasmacytoid DC numbers are comparable between age groups.
    • Fig. S2. IFN Induction in response to dsRNA transfection depends on a 5′-ppp motif in human monocytes.
    • Fig. S3. Basal IFNB expression is comparable in human monocytes from older and younger donors.
    • Fig. S4. IFN-β pretreatment is insufficient to ablate age-related differences in IFN-β secretion upon RIG-I stimulation.
    • Fig. S5. Basal levels of RIG-I, IRF3, and IRF7 protein are comparable in older and younger monocytes.
    • Fig. S6. IRF3 phosphorylation is impaired in monocytes from older donors.
    • Fig. S7. Basal TRAF3 protein levels are positively correlated with IFN-β secretion in response to RIG-I stimulation.
    • Fig. S8. Bortezomib improves antiviral responses in monocytes from older donors.
    • Fig. S9. Lack of age-related differences in IFN-β secretion in response to the cGAMP stimulation of human monocytes.
    • Fig. S10. RNA-seq highlights the differential regulation of RIG-I, IRF, and IAVrelated signaling pathways in older RIG-I–stimulated monocytes.
    • Fig. S11. Amplification-stage increase in IRF7 protein is defective in older RIGI–stimulated monocytes.
    • Fig. S12. Basal IRF8 protein and RNA levels are comparable in old and young monocytes.
    • Fig. S13. Inducible IRF8 expression is age-dependent.
    • Fig. S14. Inducible IRF8 levels are positively correlated with RIG-I–inducible IFN-β secretion.
    • Fig. S15. Bortezomib treatment of older monocytes enhances IRF8 induction upon RIG-I stimulation.
    • Fig. S16. IFN-β–stimulated gene induction and inducible protection from influenza is preserved with age in human monocytes.
    • Fig. S17. IRF8 is an IFN-inducible gene, and its induction is impaired in older monocytes.
    • Fig. S18. Lack of age-related difference in CpG DNA methylation of the IRF8 gene in human monocytes.
    • Table S1. Human donor characteristics.
    • Table S2. Differences in IFN expression in the RNA-seq data from RIG-I–stimulated older and younger monocytes.
    • Table S3. Differences in IRF expression in the RNA-seq data from RIG-I–stimulated older and younger monocytes.

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    Citation: R. D. Molony, J. T. Nguyen, Y. Kong, R. R. Montgomery, A. C. Shaw, A. Iwasaki, Aging impairs both primary and secondary RIG-I signaling for interferon induction in human monocytes. Sci. Signal. 10, eaan2392 (2017).

    © 2017 American Association for the Advancement of Science

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