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

Abstract

Adults older than 65 account for most of the deaths caused by respiratory influenza A virus (IAV) infections, but the underlying mechanisms for this susceptibility are poorly understood. IAV RNA is detected by the cytosolic sensor retinoic acid-inducible gene I (RIG-I), which induces the production of type I interferons (IFNs) that curtail the spread of the virus and promote the elimination of infected cells. We have previously identified a marked defect in the IAV-inducible secretion of type I IFNs, but not proinflammatory cytokines, in monocytes from older (>65 years) healthy human donors. We found that monocytes from older adults exhibited decreased abundance of the adaptor protein TRAF3 (tumor necrosis factor receptor-associated factor 3) because of its increased proteasomal degradation with age, thereby impairing the primary RIG-I signaling pathway for the induction of type I IFNs. We determined that monocytes from older adults also failed to effectively stimulate the production of the IFN regulatory transcription factor IRF8, which compromised IFN induction through secondary RIG-I signaling. IRF8 played a central role in IFN induction in monocytes, because knocking down IRF8 in monocytes from younger adults was sufficient to replicate the IFN defects observed in monocytes from older adults, whereas restoring IRF8 expression in older adult monocytes was sufficient to restore RIG-I-induced IFN responses. Aging thus compromises both the primary and secondary RIG-I signaling pathways that govern expression of type I IFN genes, thereby impairing antiviral resistance to IAV.

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 20-30; n=18) and older (age 65-89; n=12) healthy donors were transfected with a RIG-I–specific 5’-ppp 14-bp 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 a protein transport inhibitor cocktail 3 hours after stimulation, and at 6 hours poststimulation cells were fixed, permeabilized, and labeled for intracellular IFN-β or (C) 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. Using these plots, the frequency of (E) TNF-α+ monocytes and (F) IFN=α+ monocytes were calculated. Data are presented as means ± SEM. ** P<0.01; Student t-test.

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

(A) Monocytes from younger (n=6) and older (n=5) donors were transfected with a RIG-I–specific ligand and labeled for intracellular phosphorylated IRF3 (pIRF3) or (B) phosphorylated IRF7 (pIRF7) at the indicated timepoints (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 minutes after transfection with a RIG-I–specific ligand. Data are presented as means ± SEM. * P<0.05; Student T-test, Two-way 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) TRAF3 and β-actin abundance in older (n=6) and younger (n=6) unstimulated monocytes were 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) Older monocytes (n=12) were treated with bortezomib for 4 hours before TRAF3 and β-actin abundance were measured by Western blotting and (E) quantified by densitometry. (F) Older monocytes (n=12) were pretreated with bortezomib for 4 hours prior to 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 presented as means ± SEM. * P<0.05,** P<0.01, *** P<0.001; Paired or Unpaired Student 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. (A) Volcano plot of differentially regulated genes. Normalized values of transcripts encoding both (B) type I/III IFNs and (C) IRFs were compiled into heat maps using Microsoft Excel in order to highlight trends in 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.

Monocytes from younger (n=19) and older (n=12) human donors were (A) transfected with a RIG-I–specific ligand or (B) infected with PR8 IAV, and IRF8 expression was measured by qPCR at the indicated timepoints. Expression values for each donor were normalized to HPRT. (C, 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 (D) were quantified. Data is representative of two independent experiments. (E) Monocytes from 5 younger donors were left untreated or were treated with cyclohexamide (CHX) for 4 hours prior to mock transfection or RIG-I stimulation for 6 hours, after which IRF8 expression was quantified by qPCR. (F, G) Monocytes from younger donors were treated with an IRF8-specific siRNA or RNA-induced silencing complex (RISC)-free control for 48 hours, 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 ELISA after 12 hours of RIG-I stimulation. Data is representative of two independent experiments. (H, I) Monocytes from older donors (n=16) were transfected with an IRF8 or control lentiviral (LV) construct for 48 hours, 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 lentiviral construct for 48 hours, stimulated with a RIG-I–specific ligand for 6 hours, and IRF7 MFI was quantified by flow cytometry. Data are means ± SEM. * P<0.05,** P<0.01, ***P<0.001; Unpaired or Paired Student t-test or Two-way Repeated Measures ANOVA.

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

During the primary phase of the interferon 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 promote 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 interferon 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 interferon-α/β receptor (IFNAR) to stimulate the assembly of the interferon-stimulated gene factor 3 (ISGF3) complex, which further stimulates the production of IRF8. IRF8 cooperates with other IRF family members to amplify the production of IFNs, producing a robust antiviral interferon response. Aging impairs IRF8 induction, thereby further compromising interferon production.