Edited by: Paul W. Bland, University of Gothenburg, Sweden
Reviewed by: Franz Puttur, Imperial College London, United Kingdom; Zhengxiang He, Icahn School of Medicine at Mount Sinai, United States
This article was submitted to Mucosal Immunity, a section of the journal Frontiers in Immunology
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A decline in immune function with aging has been reported. Regulatory T cell (Treg) induction is known to decrease with age, and elucidating the underlying mechanism is important for preventing age-related diseases due to age-related chronic inflammation. In the intestine, dendritic cells (DCs) play an important role in inducing Tregs specific to oral antigens, and they efficiently induce Tregs via production of retinoic acid (RA), a vitamin A metabolite, catalyzed by the enzyme retinaldehyde dehydrogenase 2 (RALDH2). We have previously reported that in the mesenteric lymph node (MLN), a secondary lymphoid tissue in which immune responses to oral antigens are induced, four DC subsets express different levels of CD11b, CD103, and PD-L1, and we have reported that the CD11b–CD103+PD-L1high subset expresses the highest levels of the RALDH2 gene and induces Tregs
Over decades, numerous studies have revealed that aging is involved in defects in immunological functions, and a relationship between aging and chronic inflammatory conditions has been reported (
Immunosuppressive function is precisely controlled to avoid an excessive immune response to harmless substances in mucosal tissues such as intestinal tracts which are exposed to a large amount of antigens. Uncontrolled immunosuppressive function can lead to acute or chronic inflammation such as inflammatory bowel diseases (IBD). Impairment of the induction of the immunosuppressive response may be involved in age-related inflammation.
The regulatory T cell (Treg) is one of major cell types responsible for immunosuppressive function in the intestinal immune system (
Dendritic cells (DCs) are Treg inducer cells, and intestinal DCs are known to have a high ability to induce Tregs with production of retinoic acid (RA), the active vitamin A metabolite, and transforming growth factor (TGF)-β (
To examine the influence of aging on the induction of pTregs, an experimental model that can artificially induce an antigen-specific reaction without being affected by tTreg accumulation is required. In our previous study, we reported that administration of a diet containing ovalbumin (OVA) to DO11.10 mice expressing OVA-specific T cell receptors could induce the antigen-specific immune response in the intestinal immune system. In this model, a 7-day administration of the OVA diet induced Tregs in the MLN and established oral tolerance (
BALB/c mice were purchased from Charles River Laboratories Japan (Yokohama, Japan). DO11.10 mice (
The control casein (CN) diet and the egg-white (EW) diet were prepared as described previously (
RPMI 1640 (Nissui Pharmaceutical, Tokyo, Japan) containing 100 U/ml penicillin G potassium (Meiji Seika Pharma, Tokyo, Japan), 100 μg/ml streptomycin sulfate (Meiji Seika Pharma), 50 μM 2-mercaptoethanol (Tokyo Chemical Industry, Tokyo, Japan), 0.03% l-glutamine (FUJIFILM Wako Pure Chemical Corporation), and 0.2% sodium hydrogen carbonate (FUJIFILM Wako Pure Chemical Corporation) was prepared with 10% heat-inactivated fetal calf serum. For flow cytometry, anti-CD4-APC (GK1.5), anti-CD11c-APC/Cy7 (N418), purified anti-CD16/32 (93), anti-CD103-biotin (2E7), anti-CD274 (PD-L1)-PE (10F.9G2), and streptavidin-PE/Cy7 were purchased from BioLegend (San Diego, CA, United States); anti-Foxp3-PE (FJK-16s) was purchased from eBioscience (San Diego, CA, United States); and anti-CD11b-APC (M1/70) was purchased from TONBO biosciences (San Diego, CA, United States). DMSO was purchased from Sigma-Aldrich, and all-trans-retinoic acid (RA) was purchased from FUJIFILM Wako Pure Chemical Corporation. OVA323-339 peptide (OVAp) (ISQAVHAAHAEINEAGR) was purchased from Operon Biotechnologies (Tokyo, Japan).
For DC isolation, MLNs obtained from BALB/c mice were digested in 10% FCS-RPMI containing 0.5 mg/ml collagenase (FUJIFILM Wako Pure Chemical Corporation) with 10 μg/ml DNase I (Roche Diagnostics GmbH, Mannheim, Germany), and a cell suspension was obtained by filtering the digestion. From the obtained whole cells, CD11c+ cells were magnetically purified using the magnetic activated cell sorting (MACS) cell separation system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. To obtain CD11c+ cells for culture, CD11c+ purification with MACS was conducted twice to achieve high purity, and CD11c+ cells were used as DCs. For CD4+ T cell isolation, splenocytes were obtained from RAG2KO/DO11.10 mice. From whole splenocytes, CD4+ cells were separated with the MACS system. Cells were isolated from multiple mice and pooled to obtain the required number of cells. For analysis of CpG motifs methylation, MACS-enriched MLN CD11c+ cells were sorted by FACS Aria II (BD Bioscience, Franklin Lakes, NJ, United States). PBS containing 2% FCS was used as staining and washing buffer.
To analyze Foxp3 expression, MLN DCs (2 × 104 cells) from BALB/c mice and splenic CD4+ cells (2 × 105 cells) from RAG2KO/DO11.10 mice were cocultured in the presence of OVAp (100 nM) in a 96-well flat bottom plate (Corning, New York, NY, United States) in 200 μl of 10% FCS-RPMI for 72 h in a 5% CO2 humidified atmosphere at 37°C.
Total RNA was extracted from the cells using QIAShredder (QIAGEN, Hilden, Germany) and the RNeasy Mini Kit (QIAGEN). cDNA was synthesized with SuperScript VILO MasterMix (Thermo Fisher Sciences, Waltham, MA, United States). Subsequently, real-time PCR was performed to measure the relative gene expression with the QuantiTect SYBR Green PCR Kit (QIAGEN) and LightCycler (Roche Diagnostics GmbH). The relative gene expression was calculated assuming that the targeted cDNA was doubled at one cycle. The results were normalized to Gapdh gene expression as the internal control. The following primers were used: 5′-TGTCCGTCGTGGATCTGAC-3′, forward, and 5′-CCTGCTTCACCACCTTCTTG-3′, reverse, for
The cells were stained with fluorescently labeled antibodies after Fc receptor blockade by anti-CD16/32 antibody. When biotinylated antibodies were used in the staining, secondary staining was performed using streptavidin-conjugated fluorescent reagents after washing away the primary staining antibodies. To stain dead cells with propidium iodide, propidium iodide (2 μg/ml; Sigma-Aldrich) was added to samples after staining with antibodies and washed out immediately. Intracellular Foxp3 was stained using Fixation/Permeabilization Concentrate, Diluent, and Permeabilization buffer (eBioscience) according to the manufacturer’s instructions. To evaluate RALDH activity, the ALDEFLUOR assay kit (StemCell Technologies, Vancouver, Canada) was used, and RALDH activity was confirmed by comparing the control sample containing the ALDH inhibitor (N,N-diethylaminobenzaldehyde; DEAB). The fluorescence levels were measured by FACS Verse (BD Bioscience) and data analyzed using FLOWJO software (BD).
Genomic DNA was prepared from sorted cells using a PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. To analyze the methylation of CpG motifs, purified genomic DNA was modified by the bisulfite conversion reaction using an EpiMark Bisulfite Conversion Kit (New England BioLabs, Ipswich, MA, United States). The modified DNA was subjected to PCR using TaKaRa EpiTaqTM HS (for bisulfite-treated DNA) (Takara Bio, Shiga, Japan). The RALDH2 promoter region (−389 to +5 bp) was amplified by PCR from modified genomic DNA. The following primers were used: 5′-ATTTGGAATATTTAGGTAATTT-3′, forward, and 5′-ACTATATATAAACAAATATCAAA-3′, reverse, for 1st PCR; 5′-GAGTATTTATTATTTTATTTAG-3′, forward, and 5′-ACTATATATAAACAAATATCAAA-3′, reverse, for nested PCR. After amplification, the PCR products were obtained using gel electrophoresis and purified using a MinElute Gel Extraction Kit (QIAGEN). The purified PCR products were cloned into the pCR2.1 vector for sequencing. Nucleotide sequences were analyzed using Big Dye Terminator on an ABI3130xl sequencer (Applied Biosystems, Foster City, CA, United States). The methylation state for each CpG site in the PCR products was analyzed using a web-based freely available quantification tool for methylation analysis (QUMA)
Results are shown as the mean ± SD. Student’s
We used the oral tolerance model to examine the changes in the induction of Tregs with aging. We have previously reported that feeding an ovalbumin-containing diet (EW diet; egg-white diet) to DO11.10 mice for 7 days induced OVA-specific Tregs in MLNs (
Impact of aging on antigen-specific Treg induction. The proportion and absolute number of CD4+Foxp3+ cells in MLNs from young and aged RAG2KO/DO11.10 mice was analyzed. The gating strategy used to identify CD4+Foxp3+ cells is shown in
Several reports have shown that DCs showing retinaldehyde dehydrogenase (RALDH) activity promote Treg differentiation through production of RA (
Impact of aging on MLN DCs.
It has been reported that MLN DCs are a heterogenous population expressing different levels of phenotypic markers (
We hypothesized that the reduction of RALDH2 gene expression with aging was related to the changes in MLN DC subset composition. We analyzed the ratio of the subsets of MLN DCs in young or aged mice by flow cytometry. The CD11b–CD103+PD-L1high subset, which had the highest RALDH2 activity, was significantly decreased in aged mice compared with young mice, and instead, the CD11b+CD103–PD-L1int subset increased (
We compared the RALDH activity in the MLN DC subsets in young and aged mice by flow cytometry, and we found that RALDH enzyme activity was reduced in the CD11b–CD103+PD-L1high and CD11b+CD103+PD-L1high subsets (
Retinoic acid production mediated by RALDH2 in DCs affects T cells to enhance the expression of Foxp3, a transcription factor that induces differentiation into Tregs. We examined whether the ability to induce Foxp3+ T cells was reduced in MLN DCs of aged mice using DC-T cell coculture systems. The expression level of Foxp3 in T cells was analyzed by flow cytometry after coculture of MLN DCs of young or aged BALB/c mice and CD4+ T cells derived from RAG2KO/DO11.10 mouse splenocytes. A decrease in Foxp3 induction was observed in the CD4+ T cells cocultured with MLN DCs from aged mice compared with young mice (
Impact of aging on Foxp3 expression in a DC-T cell coculture. The proportion of Foxp3+ CD4+ cells after coculture of spleen CD4+ T cells from RAG2KO/DO11.10 mice and MLN DCs from young or aged BALB/c mice was analyzed. The gating strategy used to identify CD4+Foxp3+ cells is shown in
Epigenetic changes such as methylation of CpG motifs are known as factors that suppress gene expression with aging (
Analysis of methylation in CpG motifs of the MLN DC RALDH2 promoter region.
To investigate whether the influence of aging on Treg induction by EW feeding was due to a decrease in RA production, RA was administered to aged RAG2KO/DO11.10 mice and the recovery of Treg induction was examined. RA was intraperitoneally administered once per day simultaneously with the start of EW diet feeding, and the proportion of Tregs in the MLN was measured 7 days later by flow cytometry. It was observed that Treg induction tended to increase with RA administration (
Effect of RA administration on the induction of antigen-specific Tregs. The proportion of CD4+Foxp3+ cells in MLNs were analyzed. The gating strategy used to identify CD4+Foxp3+ cells is shown in
In this study, we found aging-related alterations in MLN DCs. In aged mice, the MLN DC subsets which have high RALDH gene expression and enzymatic activity in young mice, decreased in its frequencies and RALDH activity. These age-related changes were reflected in decreased RALDH2 gene expression of overall MLN DC population. These results suggested that the ability to produce RA was decreased in the intestinal immune system of aged mice. In addition, methylation of the CpG motifs in the promoter region was increased in MLN DCs of aged mice, suggesting that RA production is regulated by epigenetic changes. To date, there have been no reports showing that decreased RALDH expression in the intestinal immune organs with aging is associated with Treg induction. Treg induced in the intestinal immune system controls the immune response to oral antigens. Our results suggest that aging leads to the decreased RA production by MLN DCs, which subsequently reduces the induction of Tregs and contributes to inflamm-aging.
Several groups have reported that the ability to induce Tregs decreases with age. According to Carpentier et al., a low rate of Treg differentiation was observed 28 days after the transfer of T cells from aged C57BL/6 mice to young mice (
Numerous reports have already shown the effects of RA on the induction of Tregs (
The significant decrease in the ratio and the tendency to decrease in the numbers of CD11b–CD103+PD-L1high subset in aged mice should result in a decrease in the interaction of this DC subset with T cells, leading to decreased Foxp3+ T cell induction. We cannot deny the possibility that the MLN DC subset composition and RALDH activity changed as a result of a decrease in the ability to migrate from the lamina propria to MLNs in a subset that strongly expresses RALDH. In fact, the CD103+ subset of DCs has been reported to migrate to the MLN depending on CCR7 expression after acquiring antigen in the lamina propria (
Epigenetic modification has been reported to regulate gene expression with aging (
The decrease in Treg induction with aging can be caused not only by DC but also T cell functional decline. In fact, a decrease in immune function with aging has been reported in the acquired immune system (
Intestinal RALDH expression has been reported to depend on dietary vitamin A (
It was difficult to investigate whether the alterations in DCs observed in this study affected inflamm-aging. Even if DCs derived from aged mice are transferred into young mice, it is considered very difficult to evaluate the effect of the transferred cells since the life span of DCs is several days (
In the RA administration experiment, a tendency to promote Treg induction was observed, implying that the application of RA restored Treg induction in aged mice. Several studies have reported that RA administration has anti-inflammatory effects and enhances Treg induction. Bai et al. reported that intraperitoneal administration of RA ameliorates trinitrobenzene sulfonic acid (TNBS)-induced colitis and up-regulates Foxp3 expression in colonic tissues (
In the present study, we showed a relationship between decreased Treg induction with age and alterations in MLN DC RA production. To date, no reports have clarified the underlying mechanism of the development of inflamm-aging. Several groups have proposed theories concerning the factors involved in age-related inflammation, including: oxidative stress, DNA damage, senescence in stem cells (
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The animal study was reviewed and approved by Institutional Animal Care and Use Committee, Graduate School of Agriculture and Life Sciences, The University of Tokyo.
TT designed the study, performed the experiments, analyzed the data, and wrote the manuscript. RK contributed to the dendritic cell experiments and reviewed the manuscript. JP and TY contributed to the dendritic cell experiments. YW provided the RAG2KO/DO11.10 mice. MT and TM contributed to experiments on aging and reviewed the manuscript. KT supervised the experiments on epigenetic modification. HN-A supervised the study and reviewed the manuscript. SH designed and supervised the study and critically edited the manuscript. All authors read and approved the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thank Yimei Wang for technical assistance.
The Supplementary Material for this article can be found online at:
Gating strategy used to identify CD4+Foxp3+ cells. CD4+ cells were gated from FSC-SSC gated cells, and the proportion of Foxp3+CD4+ cells were analyzed.
Flow cytometric analysis of MLN DC subsets.
Gating strategy used to identify CD4+Foxp3+ cells in a DC-T cells coculture system. Single cells were gated, and autofluorescence+ cells (PerCP-Cy5.5 was measured as autofluorescence) were depleted from FSC-SSC gated cells. OVAp-specific (D10 TCR+) CD4+ T cells were gated, and the proportion of Foxp3+CD4+ cells were analyzed.
Gating strategy used to identify CD4+Foxp3+ cells in an RA administration experiment. Single cells were gated, and autofluorescence+ cells (FITC was measured as autofluorescence) were depleted from FSC-SSC gated cells. CD4+ T cells were gated, and the proportion of Foxp3+CD4+ cells were analyzed.
Impact of aging on T cells expressing Foxp3 in a DC-T cell coculture system.
Impact of aging on T cells expressing CCR9 in a DC-T cell coculture system.