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. 2020 Sep 1;23(9):101520. doi: 10.1016/j.isci.2020.101520

Aging-Associated Extracellular Vesicles Contain Immune Regulatory microRNAs Alleviating Hyperinflammatory State and Immune Dysfunction in the Elderly

Hirotake Tsukamoto 1,∗, Takahisa Kouwaki 1, Hiroyuki Oshiumi 1,2,∗∗
PMCID: PMC7495115  PMID: 32927264

Summary

Aging-associated changes in the immune system often lead to immune dysfunction; however, the mechanisms that underlie this phenomenon have yet to be fully elucidated. This study found that the microRNA-192 (miR-192) is an aging-associated immune regulatory microRNA whose concentration was significantly increased in aged extracellular vesicles (EVs) due to the hyperinflammatory state of aged mice. Interestingly, EV miR-192 exhibited anti-inflammatory effects on macrophages. In our aged mouse model, aging was associated with prolonged inflammation in the lung upon stimulation with inactivated influenza whole virus particles (WVP), whereas EV miR-192 alleviated the prolonged inflammation associated with aging. The hyperinflammatory state of aged mice resulted in reduced production of specific antibodies and efficacy of vaccination with WVP; however, EV miR-192 attenuated this hyperinflammatory state and improved vaccination efficacy in aged mice. Our data indicate that aged EVs constitute a negative feedback loop that alleviates aging-associated immune dysfunction.

Subject Areas: Human Physiology, Immunology, Molecular Biology, Molecular Physiology

Graphical Abstract

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Highlights

  • •

    Extracellular vesicle (EV) miR-192 is an aging-associated microRNA

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    Hyperinflammatory state in aged mice increases miR-192 levels in EVs

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    miR-192 within EVs attenuates excessive inflammation in aged mice

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    EVs containing miR-192 improve vaccination efficacy of aged mice

Human Physiology; Immunology; Molecular Biology; Molecular Physiology

Introduction

The innate immune system induces inflammatory responses upon stimulation with pathogen-associated molecular patterns (PAMPs), leading to recruitment and accumulation of myeloid and lymphoid cells that result in the activation of adaptive immune responses (Akira et al., 2006; Banchereau et al., 2000). Toll-like receptors (TLRs) are crucial to the recognition of viral and bacterial infection (Kawai and Akira, 2011). TLR7 senses viral RNA in the endosomes as a PAMP and induces the expression of type I interferon (IFN), pro-inflammatory cytokines, and chemokines, such as interleukin (IL)-6 and CCL2 (Diebold et al., 2004; Hemmi et al., 2002). CL097 and R848 are synthetic ligands for TLR7 that have been used as immune regulatory small molecules (Hemmi et al., 2002). TLR4 recognizes gram-negative bacteria by sensing lipopolysaccharide (LPS) (Medzhitov et al., 1997). The activation of TLRs leads to the maturation of dendritic cells (DCs) and is a key component in the priming of naive T cells (Dalod et al., 2014).

TLR ligands exhibit adjuvant properties during vaccination. For instance, inactivated whole virus particles of the influenza A virus (WVP) are used for vaccination against seasonal influenza and contain viral RNA with adjuvant activity in the form of TLR7 activation (Koyama et al., 2010). Monophosphoryl lipid A, which activates TLR4, is used as an adjuvant in the human papilloma virus vaccine (Garcon et al., 2007). Although vaccination is the first line of prophylaxis against infectious diseases, aging effects on the immune system can lead to immune dysfunctions, resulting in reduced vaccination efficacy in the elderly (Brodin et al., 2015; Ferrucci and Fabbri, 2018; Pinti et al., 2016).

A hyperinflammatory state has been observed in elderly humans and animals, wherein levels of IL-6 and several other pro-inflammatory cytokines in the blood are elevated (Fulop et al., 2014; Pinti et al., 2016). Inflammation itself is a necessary part of immune cell-mediated host protection, with pro-inflammatory cytokines mainly produced by innate immune cells, including macrophages, which are essential components for counteracting viral infections before the development of acquired immunity (Rose-John et al., 2017). However, the onset and termination of inflammatory responses must be tightly regulated because excessive inflammation or unbalanced production of inflammatory cytokines and chemokines can be detrimental to the organism via the amplification of tissue damage and injury and the increased susceptibility to fatal secondary infection in influenza-infected hosts (Peiris et al., 2009; Wang et al., 2014). Interestingly, the hyperinflammatory response has been reported to reduce vaccine efficacy (Fourati et al., 2016; Park et al., 2014). In contrast to pro-inflammatory cytokines, type I IFN production decreases in the elderly alongside an aging-associated decrease in intracellular TRAF3 levels (Molony et al., 2017). The production of free radical and reactive oxygen intermediates by neutrophils and macrophages is also diminished in aged mice (Gomez et al., 2005). Aging-associated changes in the immune system are readily observed, and an accumulating body of evidence has shown that the hyperinflammatory state, lower production of type I IFN, and altered functions of macrophages and neutrophils contribute to immune dysfunction in the elderly. However, the mechanisms that underlie this phenomenon have yet to be fully elucidated.

Recent studies have revealed that small extracellular vesicles (EVs) mediate intercellular communications and influence our immune system (Kouwaki et al., 2017b; Robbins and Morelli, 2014). Aging and senescence have been found to modulate EV function, but it remains unclear whether aging affects EV-mediated immune regulation. EVs consisting of 30- to 150-nm lipid bilayer vesicles are the potent systemically circulating factors that regulate immune responses, including inflammation (Colombo et al., 2014; Robbins and Morelli, 2014). These vesicles are secreted by many types of cells throughout the body for local or remote cell-to-cell communication, and they contain functional proteins and RNAs, such as microRNAs (miRNAs), which modulate cellular responses. Because of their lipid origins, EVs are readily engulfed by many different types of cells (Alexander et al., 2015; Valadi et al., 2007). Recent studies have shown that EVs deliver several immune regulatory miRNAs suited to different tasks. EV miR-155 enhances pro-inflammatory cytokine expression, and EV miR-146a attenuates inflammatory responses (Alexander et al., 2015). Circulating EVs deliver these immune regulatory miRNAs to DCs and macrophages and can fine-tune inflammatory responses (Alexander et al., 2015). We recently reported that circulating EVs with immune regulatory miRNAs control the inflammatory response of macrophages upon stimulation with the WVP, which is used for vaccination against seasonal flu (Okamoto et al., 2018), suggesting that circulating EVs also participate in the immune response to vaccines. We found that circulating EVs contained high concentration of immune regulatory miRNAs whose levels were correlated to local inflammation at the vaccination site (Miyashita et al., 2019).

In this study, we established an aging mouse model wherein aged mice exhibited lower vaccination efficacy than young mice and identified miR-192, an aging-associated miRNA present in EVs. Interestingly, circulating aging-associated EVs were crucial to the regulation of immune responses to vaccines. Our experiments indicated that the aging-associated EV miR-192 attenuated the hyperinflammatory state and improved vaccine efficacy in geriatric mice. These observations suggest a mechanism of EV-mediated control of immune dysfunction in aging.

Results

miR-192 Is an Aging-Associated miRNA in Circulating EVs

We compared miRNA profiles in serum EVs derived from young (2–3 months) and aged (14–18 months) mice to uncover aging-associated immune regulatory miRNAs in circulating EVs. Total RNAs were extracted from serum EVs of wild-type (WT), IL-6 knockout (KO), and IL-6 receptor (IL6R) KO mice Tsukamoto et al., 2017, and these were subjected to high throughput RNA sequencing (RNA-seq) analysis (Figure 1A). RNA-seq analysis revealed 15 miRNAs whose expression was specifically increased in aged WT mouse serum EVs (Figure 1B). We performed RT-qPCR to confirm our RNA-seq data and found that miR-19b, miR-322, miR-192, miR-21, and miR-181c levels in serum EVs were significantly increased in aged WT mice (Figure 1C). As some of other miRNAs were not detectable in RT-qPCR, we do not exclude the possibility that other miRNAs also increased with aging.

Figure 1.

Figure 1

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Age-Associated miRNAs in EVs

(A and B) RNAs were isolated from serum EVs of five young and aged WT, IL-6 KO, and IL6R KO mice and mixed in each group. Expression profiles of miRNAs in the pooled RNA were assessed by RNA-seq analysis (A). miRNAs with higher expression levels in aged WT mice than those in young WT mice are shown in (B) (heatmap).

(C) Serum EVs were isolated from young and aged WT and IL-6 KO mice (n = 6), and RNA was extracted from isolated EVs. The expression of indicated miRNA levels was determined by RT-qPCR. ∗p < 0.05.

(D) RAW264.7 cells were transfected with indicated miRNA mimic RNA; 36 h after transfection, cells were stimulated with R848, and mRNA expressions of Il6, Tnfa, and Ccl2 were determined by RT-qPCR (∗p < 0.05; n = 3).

(E and F) Concentrations (E) and sizes of EVs (F) in young and aged sera as determined by a NanoSight NS300.

(G) Absolute copy number of EV miR-192 as determined by RT-qPCR (∗p < 0.05; n = 5).

(H) CD63, Hsp70, and β-actin in EVs collected from sera and culture supernatant and those of whole-cell extracts were detected by western blotting with indicated Abs.

(I) EVs were collected from plasma of young and aged WT and IL-6 KO mice (n = 6). Total RNA was extracted from EVs, and miR-192 levels were determined by RT-qPCR. ∗p < 0.05. Data represent means ± SEM.

See also Figure S1.

Next, we investigated whether the aging-associated miRNAs regulate the cytokine expression. Mimic RNA for each miRNA was transfected into RAW264.7 cells, and the effect on the cytokine expression in response to a TLR ligand was investigated. Interestingly miR-192 reduced Il6 and Ccl2 mRNA levels (Figure 1D). To further clarify this point, we performed microarray analysis and found that miR-192 changed the expression of cytokines, and gene ontology and pathway analyses suggested that miR-192 was related to immune system process and cytokine signaling pathway (Figures S1A–S1C). These data imply that miR-192 plays a role in regulating cytokine responses, and thus we focused on the role of miR-192.

Concentrations of serum EVs in aged mice were comparable with those in young mice (Figure 1E). Sizes of collected EVs were around 50–200 nm, which include both exosomes and microvesicles, and no significant difference in diameters was observed between young and elderly mice (Figure 1F). Absolute copy number of miR-192 in EVs was significantly increased in aged mice (Figure 1G). We confirmed that collected EVs expressed CD63 and Hsp70, which are exosome markers (Figure 1H). Increased miR-192 levels in EVs of aged mice were also observed when EVs were collected from the plasma with anti-CD9, CD81, and CD63 antibodies (Abs), which are the markers of the exosomes (Figure 1I).

Next, we sought to uncover the mechanism underlying the increase of miR-192 in aged EVs. As pro-inflammatory cytokines are well-known to be related to aging, we investigated the effect of pro-inflammatory cytokines on the expression of miR-192. Administration of anti-IL-6 antibody (Ab), but neither anti-tumor necrosis factor (TNF)-α nor -IL-1β Ab, reduced serum EV miR-192 levels of aged mice (Figure 2A). Conversely recombinant IL-6, but neither TNF-α nor IL-1β, increased serum EV miR-192 levels in young mice (Figure 2B). In addition, IL-6 KO reduced serum EV miR-192 levels in aged mice (Figures 1C, 1G, and 1I). As previously reported Pinti et al., 2016, serum IL-6 levels in aged mice were higher than those in young mice (Figure 2C), and aged IL-6 KO mice presented with reduced levels of miR-192 comparable with those of normal young mice (Figure 1C), implicating IL-6 signaling in the increase in miR-192 observed in aged EVs. Intravenous administration of IL-6 transiently increased serum IL-6 levels and led to the increase of miR-192 levels in the serum EVs of young mice at 2 days post-IL-6 injection (Figures 2D and 2E), whereas EV miR-192 was not further increased in aged mice due to their constitutively higher level of IL-6 (Figure 2E). These data indicate that elevated IL-6 concentration causes the increase of miR-192 levels in serum EVs. It is possible that the decrease of serum IL-6 level at 2 days (Figure 2D) was caused by absorption and/or degradation of injected IL-6.

Figure 2.

Figure 2

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IL-6 Affects EV miR-192 Levels

(A) Control Ab or indicated Abs were injected into aged mice 1 and 3 days before serum harvest. EV miRNA levels were determined by RT-qPCR (∗p < 0.05; n = 6).

(B) Indicated recombinant proteins were intravenously injected into young mice; 2 days after injection, sera were collected, and EV miRNA levels were determined by RT-qPCR (∗p < 0.05; n = 6).

(C) Serum IL-6 levels in young and aged mice as determined by ELISA (∗p < 0.05; n = 10).

(D) IL-6 was injected into young mice (n = 4). One and 2 days after injection, sera were collected at indicated time points, and serum IL-6 levels were determined by ELISA (n = 4).

(E) IL-6 was injected into young and aged WT mice; 2 days after injection, serum EV miR-192 levels were assessed by RT-qPCR (∗p < 0.05; n = 4 or 5).

(F) Anti-CSF-1R was injected 5 and 2 days before EVs were collected from mouse sera. miR-192 levels in EVs were assessed by RT-qPCR (∗p < 0.05; n = 4 or 5).

(G) Splenic CD11b+ cells were collected using MACS column and stimulated with R848 in the presence or absence of IL-6 (300 ng/mL) for 1 day. EVs were collected from the cell culture medium, and miR-192 levels in EVs were assessed by RT-qPCR (∗p < 0.05; n = 5).

(H) BMMs were stimulated with mock, R848, CL097, LPS, and poly I:C in the presence and absence of 200 ng/mL of IL-6 for 48 h. EVs were collected from cell culture supernatant, and miR-192 levels in EVs were determined by RT-qPCR (∗p < 0.05; n = 3). Data represent means ± SEM.

As macrophages are the potent regulators of inflammatory response, we assessed their contribution to the increased miR-192 in EVs. Depletion of macrophages with anti-colony-stimulating factor 1 receptor (CSF-1R) Ab significantly reduced the levels of serum EV miR-192 in aged mice (Figure 2F), suggesting that macrophages are responsible for the increase of miR-192 levels in serum EVs in vivo.

In contrast to in vivo situation, IL-6 alone failed to increase miR-192 levels in EVs released from macrophages in vitro, but co-stimulation with R848, a TLR7 ligand, increased EV miR-192 levels (Figures 2G and 2H). Although CL097 or poly I:C alone failed to increase EV miR-192 levels, LPS stimulation increased miR-192 levels released from macrophages (Figure 2H). These observations imply that some kinds of stimulation with PAMPs and/or damage-associated molecular patterns, such as DNA/RNA released from host cells, are required for IL-6 to increase serum EV miR-192 levels in vivo. We do not exclude the possibility that R848 or LPS stimulation increased miR-192 levels in EVs through regulating the sensitivity to IL-6.

EV miR-192 Attenuates Pro-inflammatory Cytokine Expression

As miR-192 has the ability to suppress the cytokine expression in response to a TLR ligand (Figure 1D), we next focused on determining its activity. Bone-marrow-derived macrophages (BMMs) were transfected with miR-192 mimic RNA and then stimulated with TLR ligands, such as LPS, R848, CL097, and poly I:C. Interestingly, miR-192 mimic RNA significantly reduced the expression of Il6 and Ccl2 in response to LPS, R848, and CL097 (Figure 3A). Microarray analysis for miR-192-regulating gene expression profile in LPS-stimulated macrophages revealed that the expressions of several cytokines and chemokines were reduced by miR-192 (Figure 3B). These results were confirmed by RT-qPCR (Figure 3C). As anti-IL-6 Ab did not affect the suppression by miR-192 (Figure 3B), our data weakened the possibility that miR-192-mediated suppression of the cytokine expression was caused by reduced IL-6 expression. In general, a single miRNA targets multiple genes, and our microarray data showed that expression levels of over 100 genes were changed at least 2-fold by miR-192 (Figure S1). Time course analysis also confirmed that miR-192 attenuated the expression of Il6 and Ccl2 after R848 stimulation in both mouse RAW264.7 and human CD14+ monocyte-derived macrophages (Figures 3D and 3E). The effect on Tnfa expression was modest (Figure 3D). The IL-6 production associated with CL097 and R848 stimulation was reduced by miR-192 (Figures 3F and 3G). These data indicate that miR-192 suppresses the expression of several pro-inflammatory cytokines and chemokines.

Figure 3.

Figure 3

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miR-192-Enriched EVs Attenuate the Expression of Pro-inflammatory Cytokines

(A) BMMs were transfected with control or miR-192 mimic RNA; 36 h after transfection, cells were stimulated with LPS, R848, CL097, and poly I:C. The expression of Il6, Ccl2, and Tnfa was determined by RT-qPCR (∗p < 0.05; n = 3).

(B) BMMs were transfected with control or miR-192 mimic RNAs and then were stimulated with LPS for 24 h in the presence of anti-IL6 or control Ab. The heatmap shows the expression profiles of the genes with at least a 2-fold change in expression upon miR-192 mimic RNA transfection in the presence or absence of anti-IL-6 antibody; genes with 1.5-fold changes in expression following anti-IL-6 Ab treatment were removed.

(C–G) RAW264.7 cells and human CD14+ monocyte-derived macrophages were transfected with control and mimic miR-192 RNAs and subsequently stimulated with LPS (C), R848 (D, E, and G), and CL097 (F). The expression of each gene was assessed by RT-qPCR (C, D, and F). The IL-6 protein levels were evaluated by ELISA (F and G) (∗p < 0.05; n = 3).

(H) RNAs within EVs produced from miR-192-transfected RAW264.7 were labeled with SYTO RNA reagent, which is a membrane-permeable dye transmitting green fluorescence, and added into BMM culture at indicated concentrations. Representative histograms of fluorescent SYTO RNA+ cells in CD11b+CD64+ macrophages (left) and their frequencies in each condition (right) are shown.

(I and J) CD11b+ Gr1- macrophages were treated with control or miR-192 EVs (×1, 1 × 109 particles, or ×2, 2 × 109 particles) collected from 0, 75 (×1), and 150 (×2) μL cell culture supernatants of control- or miR-192-transfected RAW264.7 cells and then were stimulated with R848. The expression levels of miR-192, miR-101c, Il6, and Il1b were assessed by RT-qPCR (I). IL-6 protein levels in cell culture medium as determined by ELISA (J) (∗p < 0.05; n = 3).

(K and L) Control and miR-192 EVs were intravenously administrated into young (K and L) and aged mice (L). LPS (2.5 μg/mouse) was injected intraperitoneally into EV-treated young and aged mice, and serum IL-6, TNF-α, and CXCL10 protein levels were assessed by ELISA (∗p < 0.05; n = 6).

(M) Inhibitor RNAs for miR-192 and miR-101 were transfected into BMMs. Cells were treated with EVs (3 × 109 particles) collected from young and aged sera and subsequently stimulated with R848. Expression levels of Il6, Ccl2, and Tnfa were assessed by RT-qPCR (∗p < 0.05; n = 3). Data represent means ± SEM.

See also Figure S2.

It is well known that EVs deliver miRNAs to recipient cells, as demonstrated in macrophages treated with EVs that contained fluorescence-labeled RNA (Figure 3H). Thus, we next investigated whether miR-192 enclosed in EVs could exhibit the same effect as miR-192 itself did. To prepare EVs that contain miR-192 (miR-192 EVs), control mimic or miR-192 mimic RNAs were transfected into RAW264.7 cells and EVs were collected from culture supernatants (Figure S2). CD11b+ Gr1- macrophages were treated with collected EVs and then stimulated with R848. miR-192 EVs significantly increased intracellular miR-192 levels in macrophages but not the levels of other miRNAs, such as miR-101c (negative control) (Figure 3I). Interestingly, although control EVs tended to down-regulate Il6 expression with unknown mechanism, miR-192 EVs further reduced the Il6 and Il1b mRNA expression and the IL-6 protein production after R848 stimulation (Figures 3I and 3J). In addition, after LPS intraperitoneal injection, serum IL-6 levels were reduced by intravenous injection of miR-192 EV (Figure 3K). miR-192 EVs reduced serum IL-6 levels even in aged mice stimulated with LPS (Figure 3L). These data suggest that EVs deliver miR-192 to recipient cells, thereby reducing IL-6 production both in vitro and in vivo.

Because miR-192 levels were increased in aged EVs, we next investigated the effect of aged EVs on IL-6 expression in macrophages. Although young EVs collected from sera did not affect Il6 expression in macrophages stimulated with R848, aged EVs significantly reduced Il6 expression (Figure 3M). The expression of Ccl2 was also reduced by aged EVs (Figure 3M). We assessed the properties of endogenous miR-192 in aged EVs by transfecting antagomir-192 RNA, an inhibitory RNA for miR-192 (anti-miR-192), into macrophages to suppress miR-192 function before treatment with aged EVs. Anti-miR-192, but neither control RNA nor anti-miR-101 (a negative control), negated the effects of aged EVs (Figure 3M). These data indicate that miR-192 enclosed in aged EVs is transferred into macrophages, thereby suppressing the cytokine expression in recipient macrophages.

As those cytokines and chemokines were not predicted to be potential targets by miR-192 in any database, it is possible that miR-192 targets those genes indirectly. We explored this possibility by investigating whether miR-192 targeted the transcription factors for cytokines and chemokines. When cells were transfected with mimic miR-192, degradation of IκB and phosphorylation of p65 and IKKβ caused by R848 stimulation were both reduced (Figure 4A). TBK1 phosphorylation was also reduced by miR-192 (Figure 4A). In contrast, the phosphorylation of ERK and p38 was not affected by miR-192 (Figure 4B). Nuclear localization of nuclear factor (NF)-κB was decreased by miR-192 expression (Figures 4C and 4D). As NF-κB and TBK1 transcription factors induce the expression of Il6, Ccl2, and Ifnb, our data suggest that miR-192 attenuates the expression of pro-inflammatory cytokines and type I IFNs by targeting their transcription factor pathways. Previous studies have reported that miR-192 suppresses ZEB1/2 signaling molecules in epithelial cells and glomerular cells required for pro-inflammatory cytokine expression (Kim et al., 2011; Putta et al., 2012). ZEB2 knockdown reduced Il6 mRNA expression (Figures S3A and S3B). However, miR-192 failed to reduce ZEB2 protein levels in RAW264.7 cells in our experimental condition (Figure S3A).

Figure 4.

Figure 4

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miR-19 Attenuates the Activation of NF-κB and TBK1

(A–D) RAW264.7 cells were transfected with control or miR-192 mimic RNAs and then stimulated with R848. Whole-cell extracts (WCE) were subjected to SDS-PAGE, and the proteins were resolved via western blotting with indicated Abs (A and B). One hour after stimulation with R848, cells were fixed and stained with anti-NF-κB p65 Ab (red) and DAPI (blue), and the subcellular localization was observed with a confocal microscope (C). The percentages of cells with nuclear localization of NF-κB p65 are shown in (D) (∗p < 0.05; n = 3). Data represent means ± SEM.

See also Figure S3.

EV miR-192 Constitutes a Negative Feedback Loop in Aging-Associated Hyperinflammatory State

Circulating EVs can regulate immune responses after stimulation with inactivated influenza A virus particles called WVP, which are used for vaccination against flu (Okamoto et al., 2018). Thus, we next focused on the effect of aged EVs containing miR-192 on the immune responses to WVP. First, we compared WVP-induced immune responses between young and aged mice. WVP intranasal inoculation induced a transient and systemic increase in inflammatory cytokines, such as IL-6 and CXCL10, in young mice (Figure 5A). However, the higher levels of IL-6, but not CXCL10, were prolonged in aged mouse sera (Figure 5A). Consistent with higher systemic levels of IL-6 in aged mice inoculated with WVP, local expression levels of Il6, Ccl2, and Il12b in the lung were also augmented in aged mice at later time points (Figures 5B–5G), suggesting that although WVP induced inflammatory responses locally and systemically in young and old mice alike, these effects were exacerbated in aged animals.

Figure 5.

Figure 5

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Aging-Associated Changes in Immune Responses upon Vaccination

(A–G) WVPs were intranasally inoculated into young and aged mice. Serum IL-6 and CXCL10 protein levels were evaluated by ELISA (A) (∗p < 0.05; n = 9). The mRNA expression levels of Il6 (B), Ifna (C), Ccl2 (D), Il1b (E), Il12b (F), and Tnfa (G) in the lungs of young and aged mice were assessed by RT-qPCR (∗p < 0.05; n = 5).

(H and I) Total splenocytes (splenocytes) and splenic CD11c+, CD11b+, Ly6G+, CD4/8+, and CD19+ cells were isolated from young and aged mice. Cells were then stimulated with 2 μg WVP. The IL-6 (H) and TNF-α (I) protein levels in cell culture media were evaluated by ELISA (∗p < 0.05; n = 3).

(J) Splenic CD11b+ Gr1- macrophages were stimulated with 0, 1, or 2 μg WVP for 36 h, and the IL-6 and CXCL10 levels in the culture media were evaluated by ELISA (∗p < 0.05; n = 3).

(K) Splenocytes were treated with EVs (1, 3, or 6 × 109 particles) collected from young or aged mice and then stimulated with WVP. The CXCL10, IL-6, and TNF-α protein levels were evaluated by ELISA (∗p < 0.05; n = 4). Data represent means ± SEM.

Second, we examined which types of cells are responsible for prolonged pro-inflammatory cytokine expression in aged mice by isolating different types of cells from young and aged mice that were subsequently stimulated with WVP in vitro. Significantly higher amounts of IL-6 were produced by CD11b+ Gr1- macrophages isolated from aged mice when compared with those from young mice, whereas the levels of TNF-α production and CXCL10 production in aged macrophages were lower than those of their young counterparts (Figures 5H–5J). These data suggest that those macrophages are responsible for high IL-6 production in geriatric mice.

Third, we investigated whether aged EVs could control the expression of cytokines upon WVP stimulation in broad immune cell populations. Splenocytes including macrophages, DC, B cell, and T cell populations were treated with young and aged EVs and subsequently stimulated with WVP. Although young EVs reduced IL-6 production by splenocytes, aged EVs did so to a higher degree than young EVs (Figure 5K). In addition, aged EVs suppressed Il6 expression by macrophages via miR-192 (Figure 3M). These data suggest that aged EVs could attenuate macrophage-mediated pro-inflammatory cytokine expression.

Fourth, we assessed the role of EV miR-192 in immune responses to WVP inoculation. Control and miR-192 EVs were intravenously administered to young and aged mice, and after intranasal inoculation of WVP, the expression levels of miRNAs and cytokines in the lungs of EVs-administrated mice were evaluated. Although the expression levels of Il6, Il12b, Tnfa, Ifng, and Ccl2 after WVP inoculation were higher in aged mice than in young mice 60 h after WVP inoculation, cytokine and chemokine expression levels were significantly reduced by administration of miR-192 EVs (Figure 6A). We confirmed that administration of miR-192 EVs increased miR-192 levels in the lung and those in lung CD11b+ Gr-1- macrophages (Figures 6A and 6B). Consistent with the local inflammation, miR-192 EVs also abrogated prolonged systemic increase of IL-6 in aged mice (Figure 6C). The population of alveolar macrophages (F4/80+ CD11c+ SiglecF+ cells), which are tissue-resident macrophages, was dramatically decreased by intranasal administration of WVP, whereas WVP induced the recruitment and accumulation of Ly6C+ monocytes and F4/80+ CD11c− interstitial macrophages, which are differentiated from monocytes, in the lung 60 h after inoculation (Figure 6D). Accumulation and recruitment of interstitial macrophages were more apparent in aged mice (Figures 6D and S4A). miR-192 EVs significantly suppressed recruitment and accumulation of interstitial macrophages in aged mice (Figure 6D). It is possible that miR-192 EV-mediated suppression of Il12b and Tnfa expression in the lung is due to lower accumulation of interstitial macrophages, because miR-192 failed to efficiently reduce Il12b and Tnfa expression in vitro. Collectively, these data indicate that aged EVs and miR-192 EV could attenuate aging-associated prolonged cytokine expression in response to WVP inoculation in vitro and in vivo.

Figure 6.

Figure 6

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miR-192 EVs Alleviate Hyperinflammatory State in Aged Mice in Response to WVP

(A–C) Control or miR-192 EVs were intravenously injected into young and aged mice. After 14 h, WVP were intranasally administrated. Expression levels of cytokines, chemokines, and miRNAs in the lung 60 h post-vaccination were assessed by RT-qPCR (A) (n = 5). The levels of miR-192 in CD11b+ Gr-1- myeloid population isolated from lung 30 h after vaccination were assessed by RT-qPCR (B) (n = 4). Serum IL-6 levels at indicated time points were determined by ELISA (n = 5) (C). ∗p < 0.05.

(D) Control or miR-192 EVs were intravenously injected into young and aged mice. After 14 h, WVP were intranasally administrated. The frequencies of indicated myeloid populations in CD45+ cells in the lung were assessed by flow cytometry (∗p < 0.05; n = 5).

(E and F) Aged mice were treated with macrophage-depleting anti-CSF-1R or control Ab three times before and after WVP administration. Two days after WVP administration, indicated gene expression in the lung (E) and protein levels in BALF (F) were assessed by RT-qPCR and ELISA (∗p < 0.05; n = 3).

(G) At 0 and 2 days before WVP inoculation, control and anti-CSF-1R Abs were injected into aged mice. Two days after WVP inoculation, indicated population in the spleen was analyzed by flow cytometry. Absolute numbers of indicated populations are shown (∗p < 0.05; n = 3).

(H) Indicated miRNA-enriched EVs were transferred into aged mice 18 h before WVP inoculation; 40 h after WVP inoculation, serum IL-6 levels were determined by ELISA (∗p < 0.05; n = 5). Data represent means ± SEM.

See also Figures S4 and S6.

Upon depletion with anti-CSF-1R, interstitial macrophages were reduced in the lung after WVP inoculation (Figures S4Band S4C). Interestingly, anti-CSF-1R treatment significantly reduced Il6 and Ccl2 mRNA expression in the lung after WVP inoculation (Figure 6E), suggesting that interstitial macrophages are responsible for the expression of Il6 and Ccl2 in the lung. In addition, IL-6 protein levels in the BALF were also reduced by anti-CSF-1R Ab treatment (Figure 6F). These data suggest that interstitial macrophages are responsible for the augmented inflammatory responses observed in vaccinated aged mice. These observations also support that miR-192 EVs and aged EVs reduce the cytokine expression in macrophages and attenuate hyperinflammatory state in aged mice. As anti-CSF-1R treatment also reduced the number of splenic macrophages, but not those of dendritic cells, monocytes, and PMN/MDSC in the spleen (Figures 6G and S5A), we do not exclude the possibility that other types of macrophages are involved in miR-192 EV-mediated immune regulation.

To assess the effect of other miRNAs whose expression were increased with aging, we prepared EVs containing miR-19b, miR-21, and miR-322 as miR-192 and assessed the effect on WVP-induced increase of serum IL-6 in aged mice. Interestingly, miR-21 EVs as well as miR-192 EVs suppressed IL-6 levels after WVP administration (Figure 6H). This data implies that not only miR-192 EVs but also other aging-associated EVs, such as miR-21 EVs, contribute to the suppression of age-associated inflammation.

Effect of miR-192 EVs on Macrophages and Dendritic Cells

Next, we investigated whether miR-192 EVs affected the immune responses to other types of stimulation, that is, LPS-induced peritonitis model. Administration of miR-192 EVs reduced the numbers of total cells and F4/80+ macrophages accumulated in peritoneal lavage fluid in aged mice after LPS stimulation (Figures 7A–7C). Consistent with these, IL-6 level in peritoneal lavage fluid was reduced by miR-192 EV administration (Figure 7D). These data suggest that miR-192 EVs reduce the inflammatory response not only to WVP but also to other types of stimulation, such as LPS in peritonitis model.

Figure 7.

Figure 7

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miR-192 EVs Regulate the Cytokine Expression in Macrophages

(A–D) Young and aged mice were intravenously administrated with control miRNA EVs or miR-192 EVs. Twenty four hours after administration, mice were intraperitoneally injected with LPS. Cells were collected from peritoneal lavage fluid 4 h after LPS injections. Total cell number in peritoneal lavage fluid (A), representative plots for F4/80+ macrophages and Ly6G+ neutrophils (B), absolute numbers of macrophages (C left) and neutrophils (C right), and IL-6 concentration (D) in peritoneal lavage fluid are shown. Data represent means ± SEM with n = 3 or 4; ∗p < 0.05.

See also Figure S6.

To assess the effect of miR-192 EVs on the innate immune response, we investigated whether miR-192 EVs regulate the expression of MHC and co-stimulatory molecules. Stimulation with R848 increased major histocompatibility complex (MHC)-I, MHC-II, and CD86 expression of bone-marrow-derived dendritic cells (BMDCs); however, miR-192 EVs did not change their expression levels in BMDCs stimulated with or without R848 (Figures S6A and S6B). These in vitro results were supported by the fact that administration of miR-192 EVs did not affect the expression of MHC-I, MHC-II, and CD86 in CD11c+ DCs from WVP-inoculated aged mice (Figures S6C and S6D). These data suggest that miR-192 EVs attenuate the expression of cytokines but not MHC and CD86 required for antigen presentation.

Therapeutic Potential of Aging-Associated miR-192 EVs to Improve Vaccination Efficacy in the Elderly

Previous studies have shown that excessive inflammation reduced vaccine efficacy (Fourati et al., 2016; Park et al., 2014). Thus, it is expected that aged mice exhibit an attenuated response to WVP. When WVP was inoculated into young and aged mice, antigen-specific total IgG production was significantly reduced in aged mice (Figure 8A). These data indicate that our mouse model reflects reduced vaccine efficacy in elderly mice. Interestingly, IL-6 KO improved the production of specific IgG upon WVP inoculation in aged mice (Figure 8A), suggesting that IL-6-mediated inflammation in aged mice is associated with reduced vaccine efficacy by WVP. Although IL6 KO increased Ab production in aged mice (Figure 8A), IL-6 KO mice were more susceptible to viral infection than wild-type mice even if they were vaccinated (Figure S6E). This is because IL-6 itself plays protective roles during influenza A virus infection (Farsakoglu et al., 2019). These data suggest that appropriate inflammation, but not excessive inflammation, is required for efficient vaccination and protection against viral infection.

Figure 8.

Figure 8

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miR-192 EVs Improve Flu Vaccine Efficacy in Aged Mice

(A) WVP were intranasally administrated twice into WT and IL-6 KO mice. After 14 days, antigen-specific total IgG, IgG1, and IgG2c levels in sera were assessed (∗p < 0.05; n = 10).

(B) Schedule of treatment with EVs and vaccination with WVP.

(C and D) Control and miR-192 EVs were injected into young and aged mice before WVP administration as shown in (A). Mice were then infected with influenza A virus (PR8). Mouse survival (C) and body weights of survived mice (D) were recorded. Non-vaccinated (mock) versus control EV-treated aged groups (Con EV), p > 0.01 (Log rank test). Mock versus miR-192-enriched EV-treated aged groups (miR-192 EV), p < 0.01 (log rank test) (n = 10–12).

(E) Control and miR-192 EVs were injected into young and aged mice 1 day before vaccination with WVP. Abs were used to assess IgG, IgG1, and IgG2 (∗p < 0.05; n = 6–8). Data represent means ± SEM.

See also Figure S7.

As miR-192 EVs regulated pro-inflammatory responses after WVP administration in the lungs of aged mice, we expected that miR-192 EVs would improve the efficacy of vaccination with WVP in aged mice. We tested this possibility by administering control and miR-192 EVs to mice 1 day before vaccination as described in Figure 8B. Vaccinated mice were then infected with influenza A virus, and survival and their body weight loss were recorded. In young mice, vaccination prolonged the survival of virally infected mice, and miR-192 EVs did not affect survival or weight loss after viral infection (Figures 8C and 8D). We confirmed that administration of EVs itself did not affect the protective activity of WVP vaccination (Figure S6F). In aged mice, vaccination exhibited a marginal effect on protection from flu infection (Figure 8C); however, survival of vaccinated aged mice was significantly improved by administration of miR-192 EVs (Figure 8C). These data indicate a therapeutic potential of miR-192 EVs in vaccination of the elderly. When we evaluated the Th1 and Th2 responses in young and aged mice we found that Th1 response, such as IFN-γ expression, was not induced in response to WVP in aged mice, whereas Th2 response, such as IL-5 expression, was induced even in aged mice (Figure S7), suggesting that Th1 response is attenuated in aged mice. Interestingly, specific IgG and IgG2c, but not IgG1, were markedly increased by miR-192 EVs administration into aged mice (Figure 8E). As IgG2c production is known to require Th1 response, these data suggest that miR-192 EVs improved Th1 response in aged mice. This is consistent with the observation that miR-192 EVs improved vaccine efficacy. Taken together, our data indicate that aging-associated EV miR-192 alleviates the hyper-inflammatory state, leading to improved vaccination efficacy.

Discussion

Aging gives rise to pleiotropic effects on our immune system, including inflammatory responses, and a hyperinflammatory state in aged humans and animals, termed as “inflamm-aging,” partially accounts for certain age-related immune dysfunction, such as lower vaccine efficacy (Ferrucci and Fabbri, 2018; Franceschi et al., 2000; Pinti et al., 2016). It is thereby critical that we expand our knowledge about how the systemic and local inflammatory responses are modulated in old age. Here, we found that miR-192 levels in circulating EVs were increased in aged mice. Injection of IL-6 into young mice also increased EV miR-192 levels, and a shutdown of IL-6 signaling decreased EV miR-192 levels in aged mice. On the basis of our data, we conclude that the aging-associated hyperinflammatory state, especially increased IL-6 levels in sera, is a cause of increased miR-192 levels in EVs associated with aging.

Aging-associated EV miR-192 decreased the expression of several cytokines and chemokines, such as IL-6 and CCL2. Macrophages released miR-192 EVs, and depletion of macrophages reduced circulating miR-192 EV levels in aged mice, suggesting an autocrine activity of miR-192 EVs in macrophages. IL-6 is a pro-inflammatory cytokine and is known to amplify its expression (Franchimont et al., 1997; Kawano et al., 1988; Lee et al., 2012). Thus, EV miR-192 constitutes autocrine negative feedback regulation to relieve inflammation of aged individuals and buffer aging-associated hyperinflammation. The hyperinflammatory state decreased vaccination efficacy, and miR-192 EVs rescued this efficacy in aged mice. It is possible that increased miR-192 EVs' levels with aging is not sufficient to cancel the aging effect on vaccination, and thus miR-192 EVs' administration increased the efficacy of vaccination. It is expected that this negative feedback loop may help improve vaccination efficacy in elderly humans, as well.

Our microarray data revealed that several miRNA levels in EVs were increased in aged mice. Previous studies have shown that these miRNAs control inflammatory responses. For instance, miR-322 targeted mRNA of NF-κB p50 and attenuated cytokine expression in response to LPS in RAW264.7 cells (Zhang et al., 2017). In addition, miR-181c targeted 3′ UTR region of TLR4 mRNA and downregulated TLR4-mediated expression of TNF-α, IL-1β, and iNOS (Zhang et al., 2015). miR-140-5p also targeted the TLR4/MyD88 axis and alleviated inflammation in the lung injury (Yang et al., 2018). Moreover, miR-30a bound to 3′ UTR region of IRF4, thereby suppressing IL-17-associated autoimmune inflammation (Zhao et al., 2016). As miR-322, miR-181c, miR-140-5p, and miR-30a levels in EVs were increased in aged mice (Figures 1B and 1C), our data suggest that aged EVs contain several anti-inflammatory miRNAs as well as miR-192. Considering that the miR-192 inhibitor canceled the effect of aged EVs on macrophages (Figure 3M), our data indicate that miR-192 plays a role in aged EVs-mediated anti-inflammatory effects. However, we do not exclude the possibility that not only miR-192 but also other immune suppressive miRNAs are required for aged EV-mediated suppression of pro-inflammatory cytokine expression. In general, knockout studies of miRNAs are difficult because of their redundancy (Park et al., 2010). Indeed, it has been reported that there are several miR-192-encoding loci (Lim et al., 2003). Knockout of miR-192 as well as other miRNAs might be useful to further reveal the role of aging-associated EVs.

Many host factors have been reported to contribute to the aging-associated immunological abnormalities such as severe adverse events in vaccinated aged individuals (Gouin et al., 2008). However, it remains unclear which of these phenomena should be targeted to ameliorate the pathogenic features of inflammation. Here, we showed that miR-192 EVs were associated with attenuated inflammatory responses and improved vaccine efficacy in aged mice. This finding is consistent with the theory that excessive inflammation diminishes vaccination efficacy (Fourati et al., 2016; Park et al., 2014). Considering that macrophages have strong phagocytotic activity and that the priming of naive T cells is mainly mediated by DCs in vivo (Jung et al., 2002; Mempel et al., 2004), accumulation of macrophages in the lung could lead to WVP digestion and clearance of antigens, resulting in the reduced uptake of antigens by DCs necessary for the priming of naive T cells. Our study elucidated a therapeutic potential for miR-192 EVs wherein they could be used to control harmful inflammation and improve vaccine efficacy in the elderly.

miR-192 itself has pleiotropic functions. Previous studies have shown that the p53 protein regulates the expression of miR-192 and that miR-192 represses ZEB1 and ZEB2 expression (Kim et al., 2011; Putta et al., 2012). The ZEB1/2 proteins induce the expression of pro-inflammatory cytokine, such as IL-6 (Katsura et al., 2017). In this study, we found that miR-192 attenuated the activation of NF-κB. As NF-κB and ZEB1/2 are both inducers of pro-inflammatory cytokines and chemokines, we prefer the interpretation that miR-192 regulates the activation of these transcription factors, resulting in the attenuated expression of pro-inflammatory cytokines and chemokines. miR-192 EVs constitute a negative feedback loop via IL-6; however, TNF-α or IL-12 was not largely involved in the loop. Further studies are required to uncover the detailed mechanism underlying miR-192-mediated selective regulation of inflammation.

Increases in circulating miR-192 levels have also been reported in nonalcoholic fatty liver disease, hepatocellular carcinoma caused by hepatitis B virus, and patients with adenocarcinoma (Pirola et al., 2015; Song et al., 2012; Zhou et al., 2011). These diseases are associated with inflammation, and thus it is expected that an increase of miR-192 levels in EVs might be instigated by an inflammatory state not only in the elderly but also in young individuals with inflammatory diseases. It would be interesting to test whether the negative feedback loop constituted by miR-192 EVs alleviates inflammation and immune dysfunctions in these patients in the future. Recent studies reported that cellular senescence is controlled by EVs. Senescence affects the levels of an IFN-stimulated protein, IFITM3, in EVs that induces paracrine senescence, resulting in the upregulation of IFITM and downregulation of the chemokine, IL-8 (Borghesan et al., 2019). Cellular senescence is also associated with aging, and here we showed that aged EVs could downregulate pro-inflammatory cytokines. These observations indicate that circulating EVs participate in aging-associated changes in the immune system.

Limitations of the Study

This study proposes that the EVs from aged B6 mice and their components including miR-192 have immuno-modulating ability to suppress inflammatory response in macrophages in the mouse model. On the other hand, it is also important to acknowledge an incomplete understanding in their in vivo role and specific effects on immune cells other than macrophages and DC, especially in humans. It is possible that immune-modulating properties of EVs are heavily affected by their cellular sources, recipient tissue microenvironment, and hosts' baseline immunity including genetic differences. Mechanistically, cellular context-dependent target gene(s) of miR-192 are yet to be fully determined, which might be an obstacle for comprehensive understanding of the aging-associated immune defects in vivo. These limitations should be further resolved for future translational applications utilizing the microRNA-containing EVs.

Resource Availability

Lead Contact

Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Hiroyuki Oshiumi (oshiumi@kumamoto-u.ac.jp).

Materials Availability

This study did not generate new unique materials.

Data and Code Availability

All data produced or analyzed for this study are included in the published article and its supplementary information files. The accession numbers for the RNA-seq data and for the microarray data reported in this paper are DRA009054 in DDBJ database and GSE138758 in GEO, respectively.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgment

We thank Maiko Takahashi (Kamakura Techno-Science Inc.) for her invaluable assistance with 3D-Gene analyses and all our laboratory members for technical assistant and helpful discussion. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Technology (MEXT), Japan Agency for Medical Research and Development (AMED), JSPS KAKENHI No. 18K07325, the Takeda Science Foundation, and the Kumamoto University Advance Research Project A "International Research Center for Cancer and Metabolism".

Author Contributions

H.T. and H.O. designed experiments. T.K. performed flu infection assay, and H.T. performed all other experiments. H.T. and H.O. wrote the manuscript. All authors read and approved the final manuscript.

Declaration of Interests

The authors declare no competing interests.

Published: September 25, 2020

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2020.101520.

Contributor Information

Hirotake Tsukamoto, Email: htsukamo@kumamoto-u.ac.jp.

Hiroyuki Oshiumi, Email: oshiumi@kumamoto-u.ac.jp.

Supplemental Information

Document S1. Transparent Methods, Figures S1–S7 and Table S1
mmc1.pdf (3MB, pdf)

References

  1. Akira S., Uematsu S., Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006;124:783–801. doi: 10.1016/j.cell.2006.02.015. [DOI] [PubMed] [Google Scholar]
  2. Alexander M., Hu R., Runtsch M.C., Kagele D.A., Mosbruger T.L., Tolmachova T., Seabra M.C., Round J.L., Ward D.M., O'Connell R.M. Exosome-delivered microRNAs modulate the inflammatory response to endotoxin. Nat. Commun. 2015;6:7321. doi: 10.1038/ncomms8321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banchereau J., Briere F., Caux C., Davoust J., Lebecque S., Liu Y.J., Pulendran B., Palucka K. Immunobiology of dendritic cells. Annu. Rev. Immunol. 2000;18:767–811. doi: 10.1146/annurev.immunol.18.1.767. [DOI] [PubMed] [Google Scholar]
  4. Borghesan M., Fafian-Labora J., Eleftheriadou O., Carpintero-Fernandez P., Paez-Ribes M., Vizcay-Barrena G., Swisa A., Kolodkin-Gal D., Ximenez-Embun P., Lowe R. Small extracellular vesicles are key regulators of non-cell autonomous intercellular communication in senescence via the interferon protein IFITM3. Cell Rep. 2019;27:3956–3971 e3956. doi: 10.1016/j.celrep.2019.05.095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brodin P., Jojic V., Gao T., Bhattacharya S., Angel C.J., Furman D., Shen-Orr S., Dekker C.L., Swan G.E., Butte A.J. Variation in the human immune system is largely driven by non-heritable influences. Cell. 2015;160:37–47. doi: 10.1016/j.cell.2014.12.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Colombo M., Raposo G., Thery C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol. 2014;30:255–289. doi: 10.1146/annurev-cellbio-101512-122326. [DOI] [PubMed] [Google Scholar]
  7. Dalod M., Chelbi R., Malissen B., Lawrence T. Dendritic cell maturation: functional specialization through signaling specificity and transcriptional programming. EMBO J. 2014;33:1104–1116. doi: 10.1002/embj.201488027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Diebold S.S., Kaisho T., Hemmi H., Akira S., Reis e Sousa C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science. 2004;303:1529–1531. doi: 10.1126/science.1093616. [DOI] [PubMed] [Google Scholar]
  9. Farsakoglu Y., Palomino-Segura M., Latino I., Zanaga S., Chatziandreou N., Pizzagalli D.U., Rinaldi A., Bolis M., Sallusto F., Stein J.V. Influenza vaccination induces NK-Cell-Mediated type-II IFN response that regulates humoral immunity in an IL-6-dependent manner. Cell Rep. 2019;26:2307–2315 e2305. doi: 10.1016/j.celrep.2019.01.104. [DOI] [PubMed] [Google Scholar]
  10. Ferrucci L., Fabbri E. Inflammageing: chronic inflammation in ageing, cardiovascular disease, and frailty. Nat. Rev. Cardiol. 2018;15:505–522. doi: 10.1038/s41569-018-0064-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fourati S., Cristescu R., Loboda A., Talla A., Filali A., Railkar R., Schaeffer A.K., Favre D., Gagnon D., Peretz Y. Pre-vaccination inflammation and B-cell signalling predict age-related hyporesponse to hepatitis B vaccination. Nat. Commun. 2016;7:10369. doi: 10.1038/ncomms10369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Franceschi C., Bonafe M., Valensin S., Olivieri F., De Luca M., Ottaviani E., De Benedictis G. Inflamm-aging. An evolutionary perspective on immunosenescence. Ann. N. Y. Acad. Sci. 2000;908:244–254. doi: 10.1111/j.1749-6632.2000.tb06651.x. [DOI] [PubMed] [Google Scholar]
  13. Franchimont N., Rydziel S., Canalis E. Interleukin 6 is autoregulated by transcriptional mechanisms in cultures of rat osteoblastic cells. J. Clin. Invest. 1997;100:1797–1803. doi: 10.1172/JCI119707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fulop T., Le Page A., Fortin C., Witkowski J.M., Dupuis G., Larbi A. Cellular signaling in the aging immune system. Curr. Opin. Immunol. 2014;29:105–111. doi: 10.1016/j.coi.2014.05.007. [DOI] [PubMed] [Google Scholar]
  15. Garcon N., Chomez P., Van Mechelen M. GlaxoSmithKline Adjuvant Systems in vaccines: concepts, achievements and perspectives. Expert Rev. Vaccines. 2007;6:723–739. doi: 10.1586/14760584.6.5.723. [DOI] [PubMed] [Google Scholar]
  16. Gomez C.R., Boehmer E.D., Kovacs E.J. The aging innate immune system. Curr. Opin. Immunol. 2005;17:457–462. doi: 10.1016/j.coi.2005.07.013. [DOI] [PubMed] [Google Scholar]
  17. Gouin J.P., Hantsoo L., Kiecolt-Glaser J.K. Immune dysregulation and chronic stress among older adults: a review. Neuroimmunomodulation. 2008;15:251–259. doi: 10.1159/000156468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hemmi H., Kaisho T., Takeuchi O., Sato S., Sanjo H., Hoshino K., Horiuchi T., Tomizawa H., Takeda K., Akira S. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol. 2002;3:196–200. doi: 10.1038/ni758. [DOI] [PubMed] [Google Scholar]
  19. Jung S., Unutmaz D., Wong P., Sano G., De los Santos K., Sparwasser T., Wu S., Vuthoori S., Ko K., Zavala F. In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity. 2002;17:211–220. doi: 10.1016/s1074-7613(02)00365-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Katsura A., Tamura Y., Hokari S., Harada M., Morikawa M., Sakurai T., Takahashi K., Mizutani A., Nishida J., Yokoyama Y. ZEB1-regulated inflammatory phenotype in breast cancer cells. Mol. Oncol. 2017;11:1241–1262. doi: 10.1002/1878-0261.12098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kawai T., Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity. 2011;34:637–650. doi: 10.1016/j.immuni.2011.05.006. [DOI] [PubMed] [Google Scholar]
  22. Kawano M., Hirano T., Matsuda T., Taga T., Horii Y., Iwato K., Asaoku H., Tang B., Tanabe O., Tanaka H. Autocrine generation and requirement of BSF-2/IL-6 for human multiple myelomas. Nature. 1988;332:83–85. doi: 10.1038/332083a0. [DOI] [PubMed] [Google Scholar]
  23. Kim T., Veronese A., Pichiorri F., Lee T.J., Jeon Y.J., Volinia S., Pineau P., Marchio A., Palatini J., Suh S.S. p53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1 and ZEB2. J. Exp. Med. 2011;208:875–883. doi: 10.1084/jem.20110235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kouwaki T., Okamoto M., Tsukamoto H., Fukushima Y., Oshiumi H. Extracellular vesicles deliver host and virus RNA and regulate innate immune response. Int. J. Mol. Sci. 2017;18:666. doi: 10.3390/ijms18030666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Koyama S., Aoshi T., Tanimoto T., Kumagai Y., Kobiyama K., Tougan T., Sakurai K., Coban C., Horii T., Akira S. Plasmacytoid dendritic cells delineate immunogenicity of influenza vaccine subtypes. Sci. Transl. Med. 2010;2:25ra24. doi: 10.1126/scitranslmed.3000759. [DOI] [PubMed] [Google Scholar]
  26. Lee J., Nakagiri T., Oto T., Harada M., Morii E., Shintani Y., Inoue M., Iwakura Y., Miyoshi S., Okumura M. IL-6 amplifier, NF-kappaB-triggered positive feedback for IL-6 signaling, in grafts is involved in allogeneic rejection responses. J. Immunol. 2012;189:1928–1936. doi: 10.4049/jimmunol.1103613. [DOI] [PubMed] [Google Scholar]
  27. Lim L.P., Glasner M.E., Yekta S., Burge C.B., Bartel D.P. Vertebrate microRNA genes. Science. 2003;299:1540. doi: 10.1126/science.1080372. [DOI] [PubMed] [Google Scholar]
  28. Medzhitov R., Preston-Hurlburt P., Janeway C.A., Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997;388:394–397. doi: 10.1038/41131. [DOI] [PubMed] [Google Scholar]
  29. Mempel T.R., Henrickson S.E., Von Andrian U.H. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature. 2004;427:154–159. doi: 10.1038/nature02238. [DOI] [PubMed] [Google Scholar]
  30. Miyashita Y., Ishikawa K., Fukushima Y., Kouwaki T., Nakamura K., Oshiumi H. Immune-regulatory microRNA expression levels within circulating extracellular vesicles correspond with the appearance of local symptoms after seasonal flu vaccination. PLoS One. 2019;14:e0219510. doi: 10.1371/journal.pone.0219510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Molony R.D., Nguyen J.T., Kong Y., Montgomery R.R., Shaw A.C., Iwasaki A. Aging impairs both primary and secondary RIG-I signaling for interferon induction in human monocytes. Sci. Signal. 2017;10:eaan2392. doi: 10.1126/scisignal.aan2392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Okamoto M., Fukushima Y., Kouwaki T., Daito T., Kohara M., Kida H., Oshiumi H. MicroRNA-451a in extracellular, blood-resident vesicles attenuates macrophage and dendritic cell responses to influenza whole-virus vaccine. J. Biol. Chem. 2018;293:18585–18600. doi: 10.1074/jbc.RA118.003862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Park C.Y., Choi Y.S., McManus M.T. Analysis of microRNA knockouts in mice. Hum. Mol. Genet. 2010;19:R169–R175. doi: 10.1093/hmg/ddq367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Park H.L., Shim S.H., Lee E.Y., Cho W., Park S., Jeon H.J., Ahn S.Y., Kim H., Nam J.H. Obesity-induced chronic inflammation is associated with the reduced efficacy of influenza vaccine. Hum. Vaccin. Immunother. 2014;10:1181–1186. doi: 10.4161/hv.28332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Peiris J.S., Cheung C.Y., Leung C.Y., Nicholls J.M. Innate immune responses to influenza A H5N1: friend or foe? Trends Immunol. 2009;30:574–584. doi: 10.1016/j.it.2009.09.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pinti M., Appay V., Campisi J., Frasca D., Fulop T., Sauce D., Larbi A., Weinberger B., Cossarizza A. Aging of the immune system: focus on inflammation and vaccination. Eur. J. Immunol. 2016;46:2286–2301. doi: 10.1002/eji.201546178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pirola C.J., Fernandez Gianotti T., Castano G.O., Mallardi P., San Martino J., Mora Gonzalez Lopez Ledesma M., Flichman D., Mirshahi F., Sanyal A.J., Sookoian S. Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis. Gut. 2015;64:800–812. doi: 10.1136/gutjnl-2014-306996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Putta S., Lanting L., Sun G., Lawson G., Kato M., Natarajan R. Inhibiting microRNA-192 ameliorates renal fibrosis in diabetic nephropathy. J. Am. Soc. Nephrol. 2012;23:458–469. doi: 10.1681/ASN.2011050485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Robbins P.D., Morelli A.E. Regulation of immune responses by extracellular vesicles. Nat. Rev. Immunol. 2014;14:195–208. doi: 10.1038/nri3622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rose-John S., Winthrop K., Calabrese L. The role of IL-6 in host defence against infections: immunobiology and clinical implications. Nat. Rev. Rheumatol. 2017;13:399–409. doi: 10.1038/nrrheum.2017.83. [DOI] [PubMed] [Google Scholar]
  41. Song J., Bai Z., Han W., Zhang J., Meng H., Bi J., Ma X., Han S., Zhang Z. Identification of suitable reference genes for qPCR analysis of serum microRNA in gastric cancer patients. Dig. Dis. Sci. 2012;57:897–904. doi: 10.1007/s10620-011-1981-7. [DOI] [PubMed] [Google Scholar]
  42. Tsukamoto H., Fujieda K., Hirayama M., Ikeda T., Yuno A., Matsumura K., Fukuma D., Araki K., Mizuta H., Nakayama H. Soluble IL6R expressed by myeloid cells reduces tumor-specific Th1 differentiation and drives tumor progression. Cancer Res. 2017;77:2279–2291. doi: 10.1158/0008-5472.CAN-16-2446. [DOI] [PubMed] [Google Scholar]
  43. Valadi H., Ekstrom K., Bossios A., Sjostrand M., Lee J.J., Lotvall J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 2007;9:654–659. doi: 10.1038/ncb1596. [DOI] [PubMed] [Google Scholar]
  44. Wang Z., Zhang A., Wan Y., Liu X., Qiu C., Xi X., Ren Y., Wang J., Dong Y., Bao M. Early hypercytokinemia is associated with interferon-induced transmembrane protein-3 dysfunction and predictive of fatal H7N9 infection. Proc. Natl. Acad. Sci. U S A. 2014;111:769–774. doi: 10.1073/pnas.1321748111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yang Y., Liu D., Xi Y., Li J., Liu B., Li J. Upregulation of miRNA-140-5p inhibits inflammatory cytokines in acute lung injury through the MyD88/NF-kappaB signaling pathway by targeting TLR4. Exp. Ther. Med. 2018;16:3913–3920. doi: 10.3892/etm.2018.6692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhang L., Li Y.J., Wu X.Y., Hong Z., Wei W.S. MicroRNA-181c negatively regulates the inflammatory response in oxygen-glucose-deprived microglia by targeting Toll-like receptor 4. J. Neurochem. 2015;132:713–723. doi: 10.1111/jnc.13021. [DOI] [PubMed] [Google Scholar]
  47. Zhao M., Sun D., Guan Y., Wang Z., Sang D., Liu M., Pu Y., Fang X., Wang D., Huang A. Disulfiram and diphenhydramine hydrochloride upregulate miR-30a to suppress IL-17-associated autoimmune inflammation. J. Neurosci. 2016;36:9253–9266. doi: 10.1523/JNEUROSCI.4587-15.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhang K., Song F., Lu X., Chen W., Huang C., Li L., Liang D., Cao S., Dai H. MicroRNA-322 inhibits inflammatory cytokine expression and promotes cell proliferation in LPS-stimulated murine macrophages by targeting NF-kappaB1 (p50) Biosci. Rep. 2017;37 doi: 10.1042/BSR20160239. BSR20160239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zhou J., Yu L., Gao X., Hu J., Wang J., Dai Z., Wang J.F., Zhang Z., Lu S., Huang X. Plasma microRNA panel to diagnose hepatitis B virus-related hepatocellular carcinoma. J. Clin. Oncol. 2011;29:4781–4788. doi: 10.1200/JCO.2011.38.2697. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent Methods, Figures S1–S7 and Table S1
mmc1.pdf (3MB, pdf)

Data Availability Statement

All data produced or analyzed for this study are included in the published article and its supplementary information files. The accession numbers for the RNA-seq data and for the microarray data reported in this paper are DRA009054 in DDBJ database and GSE138758 in GEO, respectively.

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