Aging-related chronic inflammation is a risk factor for many human disorders through incompletely understood mechanisms. Aged mice deficient in microRNA (miRNA/miR)-146a succumb to life-shortening chronic inflammation. In this study, we report that miR-155 in T cells contributes to shortened lifespan of miR-146a−/− mice. Using single-cell RNA sequencing and flow cytometry, we found that miR-155 promotes the activation of effector T cell populations, including T follicular helper cells, and increases germinal center B cells and autoantibodies in mice aged over 15 months. Mechanistically, aerobic glycolysis genes are elevated in T cells during aging, and upon deletion of miR-146a, in a T cell miR-155-dependent manner. Finally, skewing T cell metabolism toward aerobic glycolysis by deleting mitochondrial pyruvate carrier recapitulates age-dependent T cell phenotypes observed in miR-146a−/− mice, revealing the sufficiency of metabolic reprogramming to influence immune cell functions during aging. Altogether, these data indicate that T cell–specific miRNAs play pivotal roles in regulating lifespan through their influences on inflammaging.
Chronic inflammation in the absence of infection is a commonly observed phenomenon during aging and often involves upregulation of inflammatory cytokines, elevated autoantibody titers, and impaired or altered hematopoiesis (1). This chronic, low-grade activation of the immune system upon aging, termed “inflammaging,” is implicated in the development of several age-related deleterious conditions including heart disease, neurodegeneration, autoimmunity, metabolic diseases, and cancer (2). Studies have shown that the immune system undergoes significant changes and is skewed toward an increased production of myeloid cells that are thought to drive key inflammatory processes during aging (3, 4). Additionally, the critical involvement of T and B lymphocytes in aging-related immune dysfunction was recently appreciated (5–7). However, despite the clear correlations between chronic inflammation and disease onset or decreased lifespan, both the underlying mechanisms that drive inflammaging and approaches to treat or prevent this process remain largely unknown (8). A better understanding of these mechanisms will be critical for improving both the quality and duration of life in continually expanding elderly populations.
One way in which chronic inflammation and immune dysfunction can be manifested in the elderly is through the expansion of memory CD4+ T cells with a T follicular helper (Tfh) cell phenotype (9). Tfh cells are a subset of CD4+ T cells that provide help to germinal center (GC) B cells and promote Ab production (10). Although this process is critical for defense against pathogens and the effectiveness of vaccines, improper expansion of Tfh cells can lead to the development of autoantibodies and autoimmunity, such as in the case of lupus (11). Studies have shown that the prevalence of Tfh cells increases during aging, along with the levels of autoantibodies (12–15). Although the role of these autoantibodies in the aging process remains to be clearly elucidated, they may contribute to autoimmune diseases seen in aging populations (14, 16). Thus, an increased understanding of the mechanisms that control Tfh biology as well as other aberrant T cell responses is needed to mitigate the negative impacts of autoantibody production and other inflammatory mediators during the aging process.
MicroRNAs (miRNAs) are among the critical regulatory molecules that modulate lymphocyte biology, including Tfh and B cell functions, during aging. Two well-described miRNAs, miR-146a and miR-155, can serve as an immune rheostat by exerting opposing functions (17, 18). Both miR-146a and miR-155 are induced in multiple immune cell subsets including T cells, B cells, macrophages, and dendritic cells upon activation (18–22). miR-146a serves as a negative regulator of the immune response by suppressing the expression of several proteins involved in the TLR and TCR pathways, such as TNFR-associated factor 6 (TRAF6) and IL-1R–associated kinase 1 (IRAK1) (19, 23). In contrast, miR-155 enhances the immune response by targeting negative regulators including SHIP1, SOCS1, and SOCS3 (24–26). miR-146a and miR-155 expression can be dysregulated in various inflammatory and autoimmune conditions. For instance, miR-146a was found to be expressed at lower levels in peripheral blood leukocytes of lupus patients compared with the healthy controls (27). Furthermore, mice lacking miR-146a expression experience multiple immune pathologies upon aging, such as myeloproliferation, splenomegaly, bone marrow failure, leukemia, and autoimmunity (20, 28). Interestingly, some of these phenotypes can be reversed when miR-155 is deleted in miR-146a−/− mice (20). Supporting its immune-activating roles in miR-146a–deficient mice, overexpression of miR-155 in wild-type (WT) mice led to chronic inflammation characterized by myeloproliferation, splenomegaly and bone marrow failure (29, 30). Previously, we have shown that miR-155 expression in T cells was increased in middle-aged (∼7-mo-old) miR-146a–deficient mice compared with WT controls and that T cell–specific miR-155 mediated the autoimmune phenotype during this intermediate stage of the murine lifespan (20). These findings suggest that the interplay between miR-146a and miR-155 regulates the immunopathologies associated with aging and might impact overall lifespan.
In this study, we examined survival and immunopathology in older (∼15–16-mo-old) miR-146a–deficient mice and assessed whether T cell–specific deletion of miR-155 in this setting is able to lengthen lifespan and reduce chronic inflammation. Our data reveal that the loss of miR-146a results in the expansion of Tfh cells, multiorgan autoimmunity, and bone marrow failure in aged mice, correlating with dramatically reduced survival. Importantly, T cell–specific deletion of miR-155 in miR-146a–deficient animals suppressed the autoimmune phenotype and significantly extended their lifespan. However, bone marrow phenotypes that occurred in miR-146a−/− mice, including some aspects of myeloid skewing and bone marrow hypocellularity, were not substantially impacted by the loss of T cell miR-155 at this advanced age. Mechanistically, lack of miR-146a induced the expression of aerobic glycolysis genes and overactivation of both CD4+ and CD8+ T cells in the aging immune system. Enhanced glycolysis gene expression was largely reversed upon T cell–specific deletion of miR-155, suggesting that miR-146a and miR-155 regulate the metabolic state of T cells during the aging process. Importantly, skewing T cell metabolism to aerobic glycolysis through T cell–specific deletion of the mitochondrial pyruvate carrier 1 (Mpc1) gene resulted in the expansion of activated T cells, including Tfh cells, in aging mice similar to the miR-146a−/− condition. Taken together, our work substantially expands our understanding of the critical functions performed by miR-146a and miR-155 during age-mediated immune dysfunction, including their potential to influence lifespan.
Materials and Methods
The overall objective of the controlled laboratory experiments in this study was to characterize the roles of T cell–expressed miR-155 in the process of age-related chronic inflammation. Sample sizes in experiments involving transgenic animals were determined by power analyses and through consulting with the Biostatistics Core at the University of Utah School of Medicine. Datapoints were not removed from analyses unless they meet stringent criteria determined by statistical outlier tests. The primary end point was selected to be ∼15 mo in experiments measuring survival differences between groups based on prior experience. To address sex as an experimental variable, we conducted experiments using both male and female mice and observed similar results. Experiments were repeated multiple times unless otherwise indicated and representative results are shown.
Mice described in these studies are on the C57BL/6 genetic background and were housed in the specific pathogen-free animal facility on a 12-h light/dark cycle at the University of Utah. miR-146a−/−, miR-146a−/− CD4-Cre miR-155fl/fl, and MPC1fl/fl mice were described previously (31, 32). Founder mouse strains were purchased from Jackson Laboratories. Both female and male age-matched mice were used in all experiments. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Utah.
Processing of mouse splenocytes and bone marrow
Spleens were removed from mice, homogenized using the frosted end of microscope slides, and filtered through a 40-μM filter to obtain single-cell suspensions. RBC were then lysed with RBC lysis buffer (BioLegend), and single cells were resuspended in culture media (RPMI media containing 10% FBS, penicillin, and streptomycin). To collect bone marrow cells, femurs were isolated and bone marrow was flushed using PBS. RBCs were then lysed with RBC lysis buffer, and samples were resuspended in culture medium. Organ cellularity was assessed by enumerating isolated cells using a hemocytometer.
Splenic immune landscape was analyzed using 10× Genomics single-cell transcriptomics. Briefly, CD45+ splenic lymphocytes (forward/side scatter–low) were sorted via flow cytometry as described previously (33). Representative spleens from three (young WT males) or six (15-mo-old WT, miR-146a−/−, double knockout [DKO] males) were pooled per group, and equal numbers of cells were partitioned followed by single-cell 3′ gene expression library preparation as per manufacturer’s recommendations. A 125-cycle paired-end sequencing run was performed using a HiSeq SBS kit (v4). After sequencing, data were processed using the CellRanger pipeline (10× Genomics), which showed that at least 4750 cells were sequenced, with a minimum of 116.5 million reads per sample and the 19,586 mean reads per cell across four separate runs. Data are further analyzed using Seurat single-cell genomics R package (34). Different sequencing runs were aggregated using Seurat’s built-in functions and 10 metagene dimensions to simultaneously analyze all four experimental groups. Single-cell clusters identified by the Seurat algorithm were annotated using our previously published algorithm (33) (available online at https://aekiz.shinyapps.io/CIPR/) and by performing differential expression analyses. Visualizations were generated using R programming environment and ggpubr and fgsea R packages.
ELISA for dsDNA autoantibodies and total Ab levels
Serum autoantibodies specific for dsDNA were analyzed using a commercial ELISA kit (BioVendor) according to manufacturer’s protocol. Briefly, serial dilutions of serum were added to dsDNA-coated 96-well plates. After 2 h of incubation, plates were washed and anti-dsDNA autoantibodies were spectrophotometrically detected using an HRP-conjugated anti-mouse IgG secondary Ab. A similar experimental approach was employed to quantify the total IgG levels in which anti-mouse IgG capture Abs were used for plate coating.
To measure circulating autoantibodies specific for tissue Ags, serum was collected by spinning down blood isolated via cardiac puncture at 10,000 rpm for 10 min. Target tissues including brains, kidneys, and livers were collected after whole-body perfusion with PBS. Tissues were then homogenized with a Dounce homogenizer in RIPA buffer with Protease inhibitor (Pierce). Tissues were then spun down 12,000 rpm for 10 min, and supernatant containing tissue proteome was collected. Thirty micrograms of protein was run on a 10% SDS-PAGE gel, then transferred to nitrocellulose paper. After blocking with milk, a 1:100 dilution of serum was used as primary Ab for Western blots. Following incubation of the membranes with serum, a 1:2000 dilution of an HRP-conjugated anti-mouse IgG Ab (Jackson ImmunoResearch) was used as the secondary Ab to reveal self-reactive Abs upon treatment with ECL solution.
Fluorophore-conjugated Abs were purchased from eBioscience (Thermo Fisher Scientific) or BioLegend and used to stain RBC-depleted splenocytes or bone marrow cells. The following clones were used: B220 (RA3-6B2), CD3e (142-2c11), CD4 (GK1.5), CD8a (53-6.7), CD11b (M1/70), CD44 (IM7), CD45 (30-F11), CD62L (MEL-14), CD69 (H1.2 F3), CXCR5 (L138 D7), Fas (Jo2), GL7, IgD (11-26c.2a), ICOS (7E.17G9), PD-1 (RMP1-30), and Ter119 (TER119). Cells were stained on ice in FACS buffer (PBS containing 2% FBS and 0.01% sodium azide). After staining, cells were washed two times with FACS buffer and were fixed for 15 min in 1% formalin for future analysis. Stained cells were analyzed with a BD LSRFortessa flow cytometer equipped with four lasers. Data were analyzed using FlowJo software. CD3e+CD8a+ or CD3e+CD4+ gates were used to define T cells in the analyses. Tfh cells were defined by positively gating on PD1 and CXCR5 or PD1 and ICOS within the CD3e+CD4+ T cell subset. GC B cell subset was defined as Fas+GL7+ of the B220+IgD (low) cells in the spleen. Flow cytometry gating was applied after excluding debris and doublets using the forward/side scatter data.
Survival data were analyzed using log-rank test between pairs of groups. Organ cellularity and flow cytometry data were analyzed by ANOVA with Tukey correction using GraphPad Prism 7. In single-cell RNA sequencing (scRNAseq) experiments, gene expression differences among groups were calculated using Wilcoxon test with a Benjamini–Hochberg correction for multiple testing. Gene set enrichment analysis (GSEA) was performed using fgsea R package (A. Sergushichev, manuscript posted on bioRxiv), and p values were corrected for multiple testing using the Benjamini–Hochberg method. In experiments involving two groups (such as Mpc1 knockout versus floxed control group) Student t test was used for statistical comparisons. The p values are represented by asterisks as follows: *p < 0.05, **p < 0.01, ***p < 0.001.
Loss of miR-146a results in increased mortality during aging, which is rescued by T cell–specific deletion of miR-155
Previous work by our laboratory and others have demonstrated that miR-146a is responsible for dampening chronic inflammation during aging. Furthermore, upon reaching middle age (∼7 mo), mice lacking miR-146a begin to develop chronic inflammation that could be partially reversed by deleting miR-155 in T cells (20, 35–37). To determine if miR-155 function within T cells could impact the actual lifespan of miR-146a−/− mice, we examined both female and male mice from three groups for ∼15 mo: 1) miR-155 fl/fl (WT, n = 44), 2) miR-146a−/− (miR-146a KO, n = 34), and 3) CD4-Cre miR-155 fl/fl miR-146a−/− (miR-146a KO/miR-155–T cell conditional knockout or DKO, n = 55). As expected based on previous observations (23, 36, 38), miR-146a−/− mice exhibited a significantly reduced survival frequency compared with WT controls (Fig. 1A–C). Surprisingly, deleting miR-155 from T cell subsets in these animals significantly extended their lifespan, albeit not back to WT levels (Fig. 1A–C). These observations were consistent in both female and male mice, although the effect was more pronounced in the female cohort (Fig. 1A). Consistent with the systemic inflammation and myeloproliferative disease, the loss of miR-146a resulted in a lower body weight and increased spleen mass (Fig. 1D, 1E). T cell–specific deletion of miR-155 restored the body weight back to WT levels and significantly reduced the splenomegaly observed in both male and female miR-146a−/− mice (Fig. 1D, 1E). The same trends were observed when spleen cellularity was examined (Fig. 1F). Loss of miR-146a was characterized by the dysregulation of hematopoiesis and a decline in the numbers of hematopoietic cells in the bone marrow (Fig. 1G) (23). Interestingly, deletion of miR-155 in T cells did not affect the bone marrow hypocellularity, suggesting its survival benefit is not due to a better maintenance of the hematopoietic cells in the bone marrow. We further examined the cellular composition of the bone marrow and noted a significant reduction in the numbers of B220+ B cells (Fig. 1H), Ter119+ erythroid cells (Fig. 1I), and CD11b+ myeloid cells (Fig. 1J) in aged miR-146a−/− mice compared with WT counterparts. Consistent with the bone marrow cellularity data, these phenotypes were not rescued in the DKO mice at this time point. These findings suggest that the rescue of the miR-146a−/− phenotype by the deletion of miR-155 in T cells is partial because survival is likely mediated through at least two independent miR-146a regulated pathways: 1) T cell–independent regulation of hematopoiesis and 2) T cell miR-155–dependent regulation of autoimmunity.
miR-146a and miR-155 control the systemic immune landscape during aging and regulate T cell activation states
To study how miR-146a and miR-155 control the immune response in the context of aging, we characterized the transcriptome of individual immune cells in the spleen using scRNAseq. CD45+ splenocytes were sorted via flow cytometry from young WT (n = 3) and aged (∼15 mo) WT (n = 6), miR-146a−/− (n = 6), and DKO (n = 6) male mice, pooled per each group, and subjected to scRNAseq via 10× Genomics technology. Aggregate analysis of four groups revealed 16 unique single-cell clusters across lymphoid and myeloid lineages that exhibited dynamic changes with aging and between genotypes (Fig. 2A). These single-cell clusters were annotated using differential expression analyses and with the help of an algorithm we developed that compares gene expression signatures of single-cell clusters with the gene expression data from isolated immune cells found in the Immunological Genome Project database (www.immgen.org) (33). Supporting previous findings (1), the frequency of naive T cell subsets were found to decrease with aging in WT mice whereas both CD4+ and CD8+ memory T cell subsets were elevated (Fig. 2A, 2B). The loss of miR-146a in aged mice was associated with further dynamic changes, and T cell–specific deletion of miR-155 in miR-146a−/− animals blunted some of these alterations, suggesting miR-146a and miR-155 have opposing functions within the immune system during aging (Fig. 2B). The miR-146a and miR-155–dependent changes were observed in multiple cell subsets although some of the cell populations did not differ appreciably (Fig. 2B, 2C). These findings suggest that miR-146a functions to dampen immune cell activation as a consequence of aging and that T cell miR-155 is a key mediator of many, but not all, aspects of inflammation in the absence of miR-146a.
We next performed GSEA to investigate the transcriptional activity in immune-related pathways at the single-cell level. For this analysis, we used all the cells in our dataset expressing Cd3e and Cd4 or Cd3e and Cd8a simultaneously to focus on CD4+ and CD8+ T cells, respectively. The expression of IL-2-STAT5 signaling pathway genes was significantly enriched upon aging in WT mice in both CD4+ and CD8+ T cell subsets (Fig. 2D, 2E). Genetic deletion of miR-146a resulted in a further enrichment of this pathway compared with aged WT samples, whereas T cell–specific loss of miR-155 significantly blunted this enrichment in T cells (Fig. 2D, 2E). We observed the same patterns when GSEA was performed using IL-6–STAT3 signaling and inflammatory response gene sets (obtained from Molecular Signatures Database) (39), suggesting that miR-146a suppresses cytokine signaling and inflammation in aging T cells whereas miR-155 exacerbates the inflammatory processes during aging.
The lack of miR-146a results in the expansion of Tfh cells in a miR-155–dependent manner
Next, to characterize the immune phenotype in these mice and validate our findings from scRNAseq, we analyzed splenocytes using flow cytometry. As observed in middle-aged (∼7-mo-old) mice, we found increased overall numbers of CD4+ T cells (Fig. 3A), activated CD4+ T cells (Fig. 3B, 3C, Supplemental Fig. 1A), and reduced numbers of naive CD4+ T cells (Fig. 3D) in miR-146a−/− spleens compared with WT and DKO counterparts at old age (∼15 mo). In addition, we observed increased numbers of Tfh cells in miR-146a−/− spleens that were rescued by T cell–specific deletion of miR-155 (Fig. 3E, 3F, Supplemental Fig. 1B). Interestingly, we also observed a trending increase in the overall numbers of CD8+ T cells upon deletion of miR-146a (Fig. 3G) and a significant increase in the activated subset of these cells that was rescued back to the WT levels (Fig. 3H) in the DKO group. Similarly, the reduced total numbers of naive CD8+ T cells in miR-146a−/− mice were fully rescued by the T cell–specific deletion of miR-155 (Fig. 3I). The number of GC B cells was also found to increase in miR-146a−/− spleens in a T cell miR-155–dependent manner (Fig. 3J, Supplemental Fig. 1C). These trends were also evident when the frequencies were assessed instead of total numbers (Supplemental Fig. 2) and are consistent with our scRNAseq findings. As aging was shown to be associated with increased levels of autoreactive Abs (autoantibodies) (20, 40, 41), we next quantified the anti-dsDNA Abs in the serum of these mice. Aged miR-146a−/− serum had significantly higher levels of IgG specific for anti-dsDNA compared with WT counterparts, and this increase was reversed in DKO mice (Fig. 3K). Data from our laboratory and others have shown loss of miR-146a or constitutive expression of miR-155 results in a myeloproliferative phenotype (23, 30, 38). When we examined the spleens of aged mice, we noted significantly higher numbers of CD11b+ myeloid cells in miR-146a−/− animals, which was reduced back to WT levels in DKO counterparts (Fig. 3L). These differences were comparable to the observations in middle-aged mice (20), suggesting elevated Tfh cells, GC B cells, myeloproliferation, and autoantibodies are maintained in elderly mice. Altogether, these data suggest that deleting miR-155 in T cells prevents the expansion of activated Tfh cells and autoimmunity in aged, chronically inflamed miR-146a−/− mice.
Loss of miR-146a promotes IgG Ab responses against Ags from multiple tissue types and is largely reversed by T cell–specific deletion of miR-155
Autoantibodies can be generated against various self-tissue Ags, especially in the context of aging (42). To identify if autoantibodies are raised against tissue Ags in ∼15-mo-old miR-146a−/− mice, we performed Western blots on various tissues from WT, miR-146a−/−, DKO, and Rag−/− mice. In these assays, the whole organ protein lysates were size fractionated using denaturing PAGE (SDS-PAGE), transferred to membranes, and subsequently probed with the serum collected from different groups of aged mice as the primary Ab, followed by anti-mouse IgG secondary Abs to reveal tissue proteins bound by serum autoantibodies. In these experiments, organs and serum collected from Rag−/− mice serve as negative controls because these mice cannot produce Abs owing to the lack of B and T cells (43), and β-actin ensures similar amounts of protein loading in these assays. Consistent with the anti-dsDNA autoantibody data, miR-146a−/− mouse serum exhibited increased reactivity to multiple tissue Ags in brains (Fig. 4A), kidneys (Fig. 4B), and spleens (Fig. 4C) at old age. Deletion of miR-155 in T cells reduced autoreactivity back to levels comparable to aged WT mice in most tissues. Interestingly, we did not observe a global decrease in autoantibodies against liver self-antigens in DKO serum (Fig. 4D), although a few targets were still diminished upon deletion of miR-155 in T cells, indicating that lack of miR-155 in T cells can block autoantibody production against certain liver tissue Ags. Importantly, the increase in autoantibodies in miR-146a−/− mice was not due to increases in total Ab production (Supplemental Fig. 3). Altogether, these findings suggest that loss of miR-146a during aging results in elevated levels of autoantibodies against self-tissue Ags across multiple organs, and the increased autoantibody production can be suppressed by deleting miR-155 specifically in T cells.
miR-146a−/− T cells have enhanced aerobic glycolysis, which is reversed upon loss of miR-155
T cell activation is characterized by an increased metabolic output and a highly glycolytic state (44, 45). To investigate the metabolic changes in T cells in the process of aging, we first analyzed our previously published RNAseq data obtained from splenic CD4+ T cells sorted from middle-aged (∼7-mo-old) WT and whole-body miR-146a−/−, miR-155−/−, and DKO mice (accession: GSE58373) (20). In our previous study, CD4+ T cells from miR-146a−/− mice were found to have an enhanced activation phenotype with more profound Tfh differentiation (20). When the metabolic pathways were examined in these cells, we observed a significant enrichment of the glycolysis gene set in aged miR-146a−/− CD4+ T cells compared with aged WT counterparts (Fig. 5A). Glycolysis gene signature was also enriched in miR-146a−/− T cells when compared with DKO group, suggesting that the increased glycolysis in miR-146a−/− T cells is mediated by miR-155. Supporting this notion, when CD4+ T cells from aged WT and aged DKO mice were compared with each other, the glycolysis signature was not differentially enriched, indicating the deletion of miR-155 on miR-146a−/− background reverses the increased glycolysis back to WT levels. Consistent with these findings, the expression levels of genes regulating the glycolysis pathway including AldoA, Hif1a, Hk2, and Slc2a6 (Glut6) were higher in miR-146a−/− T cells and were rescued by DKO (Fig. 5B). These data suggest that T cells in middle-aged miR-146a−/− mice have increased glycolytic activity that is promoted by miR-155, and these miRNAs have opposing functions in regulating T cell activation and metabolism in the context of aging.
We then assessed whether the increased glycolysis phenotype is maintained in older mice (∼15 mo) using our scRNAseq dataset (Fig. 2). For these analyses, we divided our dataset into CD3e+CD4+ and CD3e+CD8a+ cells, as done previously (Fig. 2D, 2E), and performed GSEA using the glycolysis gene set. Supporting our previous findings from sorted T cells, scRNAseq indicated increased levels of CD4+ T cell glycolysis with aging, a phenotype that was exacerbated on a miR-146a−/− background (Fig. 5C). CD4+ T cells from DKO mice showed reduced expression of glycolysis pathway genes compared with miR-146a−/− counterparts, although this trend was not statistically significant. Expression levels of several genes in glycolysis pathway showed an age-dependent increase in WT cells that was further enhanced in miR-146a−/− mice and subsequently reversed by DKO (Fig. 5D). Age and miR-146−/− dependent increase in glycolysis signature within CD4+ T cells was also observed in CD8+ T cells (Fig. 5E). Multiple genes in the glycolysis pathway were upregulated in aged miR-146a−/− CD8+ T cells and were subsequently rescued by concomitant deletion of miR-155 (Fig. 5F). These data suggest that T cell–specific miR-155 is important for maintaining elevated glycolysis in aged miR-146a−/− T cells, and the balance between miR-146a and miR-155 regulates T cell metabolism and inflammation during aging.
Deletion of Mpc1 in T cells results in the enrichment of activated T and Tfh cells during aging
Because our data suggest that miR-146a–deficient T cells have enhanced aerobic glycolysis that can be reversed by deleting miR-155, we next wanted to determine if skewing T cells toward aerobic glycolysis was sufficient to promote the expansion of Tfh and GC B cells in the context of aging. To address this point, we used a newly developed transgenic mouse strain in which floxed Mpc1 (Mpc1 fl/fl) is deleted only in T cells by the CD4-Cre lineage driver. Mpc1 is critical for the mitochondrial uptake of pyruvate generated in the cytoplasm as the end product of glycolysis (46, 47). Because of the inability to transfer pyruvate into the mitochondria, Mpc1 knockout animals exhibit increased aerobic glycolysis to meet the cellular energetic demand (46, 47). To study the role of aerobic glycolysis in T cell aging, we immunophenotyped splenocytes collected from T cell–specific Mpc1−/− mice and floxed controls that were aged for ∼15 mo. Skewing T cell metabolism toward aerobic glycolysis by deleting Mpc1 increased the levels of activated CD4+ T cells and reduced the levels of naive T cells (Fig. 6A). A similar increase in the activated subset and a decrease in the naive subset were also evident in CD8+ T cell compartment (Fig. 6B). When we examined Tfh markers including PD1, CXCR5, and ICOS, we observed a statistically significant increase upon T cell–specific loss of Mpc1 (Fig. 6C, Supplemental Fig. 4A). Consistent with the changes in Tfh frequency, we also observed increased proportions of GC B cells (Fig. 6D, Supplemental Fig. 4B). Importantly, unlike the aged mice, the frequency of Tfh cells were similar in young (∼8 wk old) Mpc1−/− mice (Fig. 6E). Altogether, these data suggest that skewing T cells toward aerobic glycolysis can lead to increased Tfh and GC B cell expansion, similar to miR-146a−/− mice, in the context of aging.
In the current study, we identified a novel interaction between miR-146a and miR-155 in healthy aging and longevity. Our findings, like others, demonstrate that miR-146a is critical for a normal murine lifespan, as the genetic deletion of miR-146a results in a multiorgan autoimmune response with the involvement of Tfh cells and accelerated death. Importantly, deleting miR-155 in T cells is sufficient to reverse many of the negative effects observed in miR-146a−/− animals, including increased mortality. However, the loss of T cell–specific miR-155 did not completely rescue the lifespan of miR-146a−/− mice, which could be at least in part attributed to the inability to restore bone marrow cellularity and composition. Additionally, deleting miR-146a globally is expected to have non-T cell effects that likely also contribute to shortened lifespan. Indeed, multiple studies have shown that miR-146a functions in a variety of cell types to regulate immune responses (21, 23, 36–38, 48). Thus, although T cell–expressed miR-155 strongly contributes to inflammation during aging in this model, it is not sufficient to mediate all aspects of inflammatory disease observed in miR-146a−/− mice. An interesting connection between miR-146a and miR-155 was found by studies suggesting that miR-155 expression is elevated in miR-146a–deficient T cells and can contribute to overactivation of the immune system (21, 36). Altogether, our observations in this study provide strong evidence that miR-155 in T cells can have an adverse effect on health and survival during aging, at least in settings of chronic inflammation. Furthermore, maintaining the proper balance between miR-146a and miR-155 is critical for effective immunity against infection and cancer, in which miR-155 is protective, and in the context of age-related autoimmunity, in which miR-155 promotes pathologic conditions and impacts lifespan.
The essential roles of miRNAs in regulating T cell differentiation and function have been clearly established by prior work (49–56). Our study reveals an interesting link between the miRNA regulatory networks and cellular metabolism in shaping T cell responses in the context of aging and autoimmunity. Cellular metabolism is increasingly appreciated to be critical not only for mediating immune effector function but also for determining immune cell fate and activation states (57–60). Previously, we identified increased glycolysis in macrophages deficient in miR-146a due to increased TRAF6 and mTOR signaling (36). In this study, we provide evidence that miR-146a–deficient T cells in aged mice also have enhanced aerobic glycolysis compared with WT counterparts and that the removal of miR-155 can reverse this phenotype, suggesting that the metabolic state of T cells is a critical determinant in age-mediated inflammation. Our data are consistent with studies in cancer and nonhematopoietic cells, where miR-155 was found to promote glycolysis through mechanisms involving targeting of C/EBPβ, HDAC4, SOCS1, and PIK3R1 (61–63). Future work will explore whether miR-155-mediated promotion of glycolysis in T cells during aging occurs through these direct targets or via a unique mechanism that has not yet been described.
Several studies have recently shown that cellular metabolism influences T cell differentiation and effector responses (58, 59, 64–67). In the context of Tfh biology, metabolism plays a complex role in regulating the cellular phenotype. Some studies suggested that the Tfh master transcription factor Bcl6 represses the expression of hexokinase 2 (Hk2) and other key metabolic genes and that Tfh cells exhibit lower glycolytic activity compared with Th1 cells during infection (68, 69). On the contrary, others reported higher levels of glycolysis in Tfh cells compared with non-Tfh counterparts, and overexpressing the glucose transporter Slc2a1 on T cells was sufficient to increase steady-state Tfh and GC B cell numbers (70, 71). This discrepancy between different studies may be explained in part by the observation that autoreactive Tfh cells were highly sensitive to glycolysis inhibition whereas virus-induced Tfh cells remained unimpacted, indicating that the inflammatory context matters in Tfh biology and increased glycolysis is critical for the development of autoreactive Tfh cells (71). Our observations are in accordance with these latter findings and suggest that miR-146a and miR-155 controls the development of autoreactive Tfh cells through regulating glycolysis. Although it is widely accepted that activated T cells require higher glycolytic activity to support proliferation and effector cytokine production (65, 72, 73), our data demonstrate that skewing T cell metabolism toward aerobic glycolysis by deleting Mpc1 is sufficient to promote their activation in the context of age-mediated inflammation. This is also consistent with a recent study that showed that fissed punctate mitochondria favoring aerobic glycolysis lead to the development of effector T cells in bacterial infection models (74). Our study suggests that increased aerobic glycolysis in T cells could potentially increase morbidity in the elderly, and future studies will continue to investigate Mpc1 and metabolism-altering miRNAs in the context of aging and longevity.
In conclusion, our data highlight the importance of maintaining proper miRNA regulation in immune cells to promote healthy aging and long-term survival. Specifically, interventions targeting miR-155 expression and function in T cells may have the potential to prevent comorbidities of aging, extend lifespan, and improve the quality of life in the elderly, at least in some circumstances. Further preclinical and clinical studies will be essential to realize the potential of manipulating miRNAs in the immune system to tackle age-related disease.
We thank the University of Utah High Throughput Genomics (Opal Allen, Brian Dalley) and Bioinformatics (Chris Stubben, Chris Conley) core facilities for the help with RNA sequencing and scRNAseq data analysis. We would also like to thank the University of Utah Flow Cytometry core facility (James Marvin) for assistance with flow cytometric analysis and cell sorting.
This work was supported by National Institutes of Health (NIH) National Institutes of Aging Grant R01AG047956 and NIH Grant R01AI123106 (to R.M.O.), National Cancer Institute Grant R01CA166450 and National Institute of Allergy and Infectious Diseases (NIAID) Grant R21AI132869 (to D.S.R.), and NIAID Training Grants T32 AI055434 (to A.G.R.) and T32 AI138945-1 (to K.M.B.).
The sequence data presented in this article have been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE138222) under accession number GSE138222.
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.