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Reversal of epigenetic aging and immunosenescent trends in humans
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Download the PDF here (09/28/19)
https://onlinelibrary.wiley.com/doi/full/10.1111/acel.13028
First published: 08 September 2019
"Although, on average, trial volunteer epigenetic ages (EAs) were lower than their chronological ages (As) at baseline [(EA-A)0 < 0, Table 1], epigenetic age was nevertheless significantly decreased by treatment based on the results of all four epigenetic clocks (Figure 5a-d), with a mean change in EA-A after 12 months of about 2.5 years
Although epigenetic age does not measure all features of aging and is not synonymous with aging itself, it is the most accurate measure of biological age and age-related disease risk available today. This justifies the use of epigenetic clocks to estimate the effectiveness of putative aging interventions on a practical timescale. The present study strongly supports this approach, having demonstrated regression of epigenetic age with high statistical significance even in a one-year pilot trial involving only 9 volunteers. However, it will be necessary to verify the present results by replicating them in an appropriately powered follow-up study. Although much more remains to be done, the general prospects for meaningful amelioration of human aging appear to be remarkably promising.
For these reasons, we conducted what may be the first human clinical trial designed to reverse aspects of human aging, the TRIIM (Thymus Regeneration, Immunorestoration, and Insulin Mitigation) trial, in 2015-2017. The purpose of the TRIIM trial was to investigate the possibility of using recombinant human growth hormone (rhGH) to prevent or reverse signs of immunosenescence in a population of 51- to 65-year-old putatively healthy men, which represents the age range that just precedes the collapse of the TCR repertoire. rhGH was used based on prior evidence that growth hormone (GH) has thymotrophic and immune reconstituting effects in animals (Kelley et al., 1986) and human HIV patients (Napolitano et al., 2008; Plana et al., 2011). Because GH-induced hyperinsulinemia (Marcus et al., 1990) is undesirable and might affect thymic regeneration and immunological reconstitution, we combined rhGH with both dehydroepiandrosterone (DHEA) and metformin in an attempt to limit the "diabetogenic" effect of GH (Fahy, 2003, 2010; Weiss, Villareal, Fontana, Han, & Holloszy, 2011). DHEA has many effects, in both men and women, that oppose deleterious effects of normal aging (Cappola et al., 2009; Forti et al., 2012; Shufelt et al., 2010; Weiss et al., 2011). Metformin is a powerful calorie restriction mimetic in aging mice (Dhahbi, Mote, Fahy, & Spindler, 2005) and has been proposed as a candidate for slowing aging in humans (Barzilai, Crandall, Kritchevsky, & Espeland, 2016). Neither DHEA (Riley, Fitzmaurice, & Regelson, 1990) nor metformin are known to have any thymotrophic effects of their own."
Abstract
Epigenetic "clocks" can now surpass chronological age in accuracy for estimating biological age. Here, we use four such age estimators to show that epigenetic aging can be reversed in humans. Using a protocol intended to regenerate the thymus, we observed protective immunological changes, improved risk indices for many age-related diseases, and a mean epigenetic age approximately 1.5 years less than baseline after 1 year of treatment (-2.5-year change compared to no treatment at the end of the study). The rate of epigenetic aging reversal relative to chronological age accelerated from -1.6 year/year from 0-9 month to -6.5 year/year from 9-12 month. The GrimAge predictor of human morbidity and mortality showed a 2-year decrease in epigenetic vs. chronological age that persisted six months after discontinuing treatment. This is to our knowledge the first report of an increase, based on an epigenetic age estimator, in predicted human lifespan by means of a currently accessible aging intervention.
1 INTRODUCTION
Population aging is an increasingly important problem in developed countries, bringing with it a host of medical, social, economic, political, and psychological problems (Rae et al., 2010). Over the last several years, many biomedical approaches to ameliorating aging have been investigated in animal models, and some of these seem able to reverse general aspects of aging in adult mammals based on a variety of physiological measurements (Das et al., 2018; Ocampo et al., 2016; Zhang et al., 2017). However, to date, evidence that systemic aging can be reversed has not been substantiated by determinations of epigenetic age, which can now provide a simple but compelling indication of biological as opposed to chronological age (Horvath & Raj, 2018; Jylhava, Pedersen, & Hagg, 2017). In addition, there is a need to specifically address immunosenescence stemming from thymic involution (Bodey, Bodey, Siegel, & Kaiser, 1997). Thymic involution leads to the depletion of critical immune cell populations (Arnold, Wolf, Brunner, Herndler-Brandstetter, & Grubeck-Loebenstein, 2011), resulting in a collapse of the T-cell receptor (TCR) repertoire in humans after the age of ~63 (Naylor et al., 2005), and is linked to age-related increases in cancer incidence (Falci et al., 2013), infectious disease (Ventevogel & Sempowski, 2013), autoimmune conditions (Goronzy & Weyand, 2003), generalized inflammation (Goronzy & Weyand, 2003), atherosclerosis (Dai, Zhang, Wang, Wu, & Liang, 2018), and all-cause mortality (Fernando-Martinez et al., 2013; Roberts-Thomson, Whittingham, Youngschaiyud, & Mackay, 1974; Strindhall et al., 2007). In contrast, maintained immune function is seen in centenarians (Strindhall et al., 2007). Although thymic function in aging also depends on the supply of T-cell progenitors from the bone marrow, which declines in relation to the output of myeloid HSCs with age (Akunuru & Geiger, 2016), the net number of lymphoid precursors does not change with age (Montecino-Rodriguez et al., 2019), and migration of T-cell precursors from the bone marrow also appears to depend on thymic function (Haar, Taubenberger, Doane, & Kenyon, 1989).
For these reasons, we conducted what may be the first human clinical trial designed to reverse aspects of human aging, the TRIIM (Thymus Regeneration, Immunorestoration, and Insulin Mitigation) trial, in 2015-2017. The purpose of the TRIIM trial was to investigate the possibility of using recombinant human growth hormone (rhGH) to prevent or reverse signs of immunosenescence in a population of 51- to 65-year-old putatively healthy men, which represents the age range that just precedes the collapse of the TCR repertoire. rhGH was used based on prior evidence that growth hormone (GH) has thymotrophic and immune reconstituting effects in animals (Kelley et al., 1986) and human HIV patients (Napolitano et al., 2008; Plana et al., 2011). Because GH-induced hyperinsulinemia (Marcus et al., 1990) is undesirable and might affect thymic regeneration and immunological reconstitution, we combined rhGH with both dehydroepiandrosterone (DHEA) and metformin in an attempt to limit the "diabetogenic" effect of GH (Fahy, 2003, 2010; Weiss, Villareal, Fontana, Han, & Holloszy, 2011). DHEA has many effects, in both men and women, that oppose deleterious effects of normal aging (Cappola et al., 2009; Forti et al., 2012; Shufelt et al., 2010; Weiss et al., 2011). Metformin is a powerful calorie restriction mimetic in aging mice (Dhahbi, Mote, Fahy, & Spindler, 2005) and has been proposed as a candidate for slowing aging in humans (Barzilai, Crandall, Kritchevsky, & Espeland, 2016). Neither DHEA (Riley, Fitzmaurice, & Regelson, 1990) nor metformin are known to have any thymotrophic effects of their own.
2 RESULTS
2.1 Treatment safety and side effects
A primary concern in this study was whether increased levels of a mitogen (IGF-1) might exacerbate cancerous or precancerous foci in the prostate. Both of these changes should be detectable by measuring PSA or percent free PSA levels. However, PSA, percent free PSA, and the ratio of PSA to percent free PSA, an overall index of prostate cancer risk, improved significantly by day 15 of treatment and remained favorably altered to the end of 12 months (Figure 1a-c). A brief spike in PSA at 6 months in two volunteers was rapidly reversed and, after volunteer consultation, was interpreted as reflecting sexual activity close to the time of PSA testing. No change in testosterone levels was observed.
Another significant concern was whether augmenting immune activity might exacerbate age-related inflammation. However, CRP declined with treatment, the decline reaching statistical significance by 9-12 months (Figure 1d). The pro-inflammatory cytokine, IL-6, did not change (Figure 1e).
No remarkable changes were noted in serum albumin, lipids, hemoglobin, hematocrit, platelet count, electrolytes, and hepatic enzymes (Figure 1f). Insulin levels were in general adequately controlled by co-administration of DHEA and metformin (Figure 1g) (although one outlier increased mean insulin at 12 months), and glucose levels did not change (Figure 1h). Finally, estimated glomerular filtration rates (eGFR), which are relevant to the potential for lactic acidosis with metformin as well as to treatment efficacy, showed a statistically significant improvement after 9-12 months (with a trend toward improvement at 18 months as well) (Figure 1i). Side effects were mild, typical of rhGH administration, and did not require dosing modification except in two cases. Side effects included arthralgias (2 cases), anxiety (1 case), carpal tunnel syndrome (1 case), fluid retention (1 case), mild gynecomastia (1 case), and muscle soreness (1 case). One trial volunteer was removed from the study after approximately one month due to self-reported bradycardia, which preceded the trial, and belated admission of a strong familial history of cancer.
2.2 Thymic and bone marrow regenerative responses
Obvious qualitative improvements in thymic MRI density were observed and are illustrated in Figure 2. Quantitatively, the overall increase in the thymic fat-free fraction (TFFF) was significant at the p = 8.57 x 10-17 level based on linear mixed-model analysis, implying a restoration of thymic functional mass. The improvements were significant in 7 of 9 volunteers (Figure 3a-c). Two volunteers had abnormally low levels of thymic fat (high TFFF) at baseline, and their TFFFs did not significantly improve with treatment (peak relative changes of +9.6% (p > .3) and +12.4% (p > .2); Figure 3b). Their lack of response was not age-dependent. Instead, improvement in TFFF was dependent upon baseline TFFF, regardless of baseline age (Figure 3c).
By comparison, sternal BMFFF increased to a much lesser degree, but with such consistency (Figure 3d) as to reach high statistical significance (Figure 3e; p < .001 for single-point comparison, or p = 9.5 x 10-12 for formal linear mixed-model analysis). Bone marrow, similar to thymus, showed a pattern of increased BMFFF with increased baseline fat content, but the details of the pattern were different (Figure 3f). This difference plus the more robust replacement of thymic vs. bone marrow fat is consistent both with a specific reversal of thymic involution rather than generalized regression of body fat owing to GH administration and with possible stimulation of bone marrow T-cell progenitor production by GH (French et al., 2002; Hanley, Napolitano, & McCune, 2005).
2.3 Immune cell subset and cytokine changes
Analysis of CyTOF-defined immune cell populations revealed the most robust changes to be decreases in total and CD38-positive monocytes (Figure 4a, b) and resulting increases in the lymphocyte-to-monocyte ratio (LMR) (Figure 4c). Linear mixed-model correlation analysis also showed a significant correlation after 9 and 12 months of treatment between TFFF and the reduction in CD38+ monocyte percentage (r2 = .59, p = .01) as well as a correlation between TFFF and the ratio of lymphocytes to CD38+ monocytes (r2 = .55, p = .018). Similar trends were seen for total monocytes vs. TFFF and for the overall LMR vs. TFFF (respectively, r2 = .40, p = .039, and r2 = .45, p = .019) when correlated across all treatment times (0, 9, and 12 months). The changes in mean monocyte populations persisted 6 months after discontinuation of treatment (p = .012 for normalized CD38+ monocytes and p = .022 for normalized total monocytes: Table S1), and the increase in LMR remained highly significant at 18 months as well.
3 DISCUSSION
The TRIIM trial was designed to investigate the possibility of thymus regeneration and reversion of immunosenescent trends in healthy aging men while minimizing side effects and any possible risks. Our results support the feasibility of this goal but unexpectedly also bring to light robust evidence that regression of multiple aspects and biomarkers of aging is possible in man. These two observations may be related.
Thymus regeneration and reactivation by growth hormone administration have been established in aging rats and dogs by restoration of youthful thymic histology (Goff, Roth, Arp, & al., e., 1987; Kelley et al., 1986) and by reversal of age-related immune deficits (Kelley et al., 1986). In humans, the existence of surviving thymic tissue after the age of about 54, which is required for successful thymus regeneration in older individuals, has been questioned (Simanovsky, Hiller, Loubashevsky, & Rozovsky, 2012). Available reports indicating increased thymic CT density and immunological improvements induced by rhGH in HIV patients (Napolitano et al., 2008; Plana et al., 2011), whose thymi are physiologically unusual (McCune et al., 1998), are silent on whether regeneration was observed in individuals over the age of 50. The present study now establishes highly significant evidence of thymic regeneration in normal aging men accompanied by improvements in a variety of disease risk factors and age-related immunological parameters as well as significant correlations between TFFF and favorable changes in monocyte percentages and the LMR, independent of age up to the age of 65 at the onset of treatment. These observations are consistent with the known ability of growth hormone to stimulate hematopoiesis and thymic epithelial cell proliferation (Savino, 2007). Our finding of an increase in FGF-21 levels after 12 months of treatment suggests that thymic regeneration by the present treatment may be mediated in part by this cytokine (Youm, Horvath, Mangelsdorf, Kliewer, & Dixit, 2016), which we believe is a novel finding.
We have not found previous studies associating rhGH administration with an increased LMR or reduced monocyte levels, whereas DHEA administration may actually increase monocyte levels(Khorram, Vu, & Yen, 1997). The mechanisms involved are not clear, but the unexpected effect of our treatment on the LMR may be of considerable significance, for two reasons.
First, higher LMRs are associated with better prognoses for a variety of leading sources of human mortality, including at least 8 types of cancer (e.g., prostate cancer; Caglayan et al., 2019), atherosclerosis (Gong et al., 2018), cardiovascular disease (Ji et al., 2017), and stroke (Ren, Liu, Wang, & Gao, 2017)], and are also associated with less generalized inflammation (Gong et al., 2018). Protection from cardiovascular disease has been associated with an LMR above 5 (Gong et al., 2018). In our study, mean volunteer LMRs were below 5 at baseline but were well above 5 at the end of treatment and 6 months after the end of treatment.
Second, the great majority of monocytes are CD38-positive. CD38 is an NADase ectoenzyme and a degrader of the NAD+ precursor, nicotinamide mononucleotide, and increased CD38 expression with age appears to be the primary cause of age-related tissue NAD+ depletion in mice and most likely in man as well (Camacho-Pereira et al., 2016). Induction of CD38 with aging has been proposed to be driven by age-related inflammation, and to originate in inflammatory cells residing in tissues (Camacho-Pereira et al., 2016), which in principle might include monocytes. Although many other immune cells express CD38, we did not detect any other CD38+ immune cell population that declined in response to thymus regeneration treatment, suggesting that monocytes may be of particular significance. Our observation of a decline in CRP in combination with reduced monocyte levels therefore suggests the possibility of an increase in tissue NAD+ levels. The mechanism by which CRP was reduced is not clear, but reactivation of negative selection in the thymus could in theory reduce autoimmune-related inflammation; clearance of pro-inflammatory viruses or senescent somatic cells could also be involved. Given the relationship between age-related tissue NAD+ depletion and the onset of aging phenotypes (Das et al., 2018; Poljsak & Milisav, 2016), an increased tissue NAD+ content could be connected to our observations of reversed epigenetic aging, to the general association of higher LMRs with better health, and to previous observations linking thymus transplantation to regression of various nonimmunological aging processes (Fabris, Mocchegiani, Muzzioli, & Provinciali, 1988). Consistent with this possibility, we observed that strong declines in total and CD38+ monocytes, increases in LMR, and reduced GrimAge all persisted for 6 months following discontinuation of treatment.
Treatment-induced declines in PD-1+ CD8 T cells are consistent with the possibility that thymus regeneration treatment epigenetically reprograms "exhausted" CD8 T cells (TEX), a recently characterized distinct PD-1+ CD8 population (Khan et al., 2019). These cells are of great interest in part because PD-1 is an immune checkpoint molecule that inhibits T-cell proliferative responses (Henson, Macaulay, Franzese, & Akbar, 2012) and is involved in cancer evasion of immune control, leading to intensive efforts to develop pharmacological immune checkpoint inhibitors that target PD-1 (Khan et al., 2019). Blocking PD-1 signaling improves proliferation of "senescent" human CD-8 cells (Henson, Macaulay, Riddell, Nunn, & Akbar, 2015). Mice, too, develop a subpopulation of exhausted CD8 cells with aging that are PD-1+ and Tim-3+ (Lee et al., 2016). Therefore, reduction of PD-1+ CD8 T cells with thymus regeneration treatment likely represents a significant improvement in immune status.
Treatment induced increases in naïve CD4 and naïve CD8 T cells were relatively small compared to changes reported in rhGH treated HIV patients, but our volunteer population was pre-immunosenescent and not depleted of naïve CD4 and naïve CD8 T cells at baseline. Positive responses also occurred despite potential complications caused by lymph node aging (Thompson et al., 2019). Therefore, the small increases observed in these cells and in CD4 T-cell RTEs are consistent with the ultimate goal of preventing or reversing the normal age-related collapse of the TCR repertoire at ages just above those of our study population (Naylor et al., 2005).
There may be both immunological and non-immunological mechanisms of epigenetic aging reversal. GH, DHEA, and metformin have unique effects that are in opposition to aging, and it is possible that the specific combination of these agents activates a broad enough range of therapeutic pathways to account for the previously unpredictable reversal of epigenetic aging, even independently of the immunological markers we have measured.
In this regard, it must be pointed out that GH and IGF-1 can also have pro-aging effects and that most gerontologists therefore favor reducing rather than increasing the levels of these factors (Longo et al., 2015). However, most past studies of aging and GH/IGF-1 are confounded by the use of mutations that affect the developmental programming of aging, which is not necessarily relevant to nonmutant adults. For example, such mutations in mice alter the normal innervation of the hypothalamus during brain development and prevent the hypothalamic inflammation in the adult (Sadagurski et al., 2015). Hypothalamic inflammation may program adult body-wide aging in nonmutants (Zhang et al., 2017), but it seems unlikely that lowering IGF-1 in normal non-mutant adults can provide the same protection. A second problem with past studies is a general failure to uncouple GH/IGF-1 signaling from lifelong changes in insulin signaling. Human longevity seems more consistently linked to insulin sensitivity than to IGF-1 levels, and the effects of IGF-1 on human longevity are confounded by its inverse proportionality to insulin sensitivity (Vitale, Pellegrino, Vollery, & Hofland, 2019). We therefore believe our approach of increasing GH/IGF-1 for a limited time in the more natural context of elevated DHEA while maximizing insulin sensitivity is justified, particularly in view of the positive role of GH and IGF-1 in immune maintenance, the role of immune maintenance in the retardation of aging (Fabris et al., 1988), and our present results.
Whatever the mechanism of epigenetic age reversal may be, the four selected epigenetic clocks, despite measuring somewhat different features of aging and correlating differently with blood composition and leukocyte telomere length, all showed significant regression of epigenetic age. There was also a marked acceleration of epigenetic aging reversal after 9 months of treatment. The implications of the latter observation remain to be explored. Further, although epigenetic aging reversal appeared to partially regress following discontinuation of treatment according to some epigenetic clocks, this was not true for the GrimAge clock, which best predicts human life expectancy and health span (Lu et al., 2019). It remains to be seen whether follow-up measurements of epigenetic aging using the GrimAge clock will show a persistent 2-year gain in predicted life expectancy or a gradual loss of increased life expectancy compared to baseline. In the latter event, it will be interesting to determine whether repetition or prolongation of the trial treatment might restore or further augment the predicted lifespan gain.
Although epigenetic age does not measure all features of aging and is not synonymous with aging itself, it is the most accurate measure of biological age and age-related disease risk available today. This justifies the use of epigenetic clocks to estimate the effectiveness of putative aging interventions on a practical timescale. The present study strongly supports this approach, having demonstrated regression of epigenetic age with high statistical significance even in a one-year pilot trial involving only 9 volunteers. However, it will be necessary to verify the present results by replicating them in an appropriately powered follow-up study. Although much more remains to be done, the general prospects for meaningful amelioration of human aging appear to be remarkably promising.
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