Intersection of immunometabolism and immunosenescence during aging

Abstract

Aging is associated with the highest risk for morbidity and mortality to chronic or metabolic diseases, which are present in fifty percent of the elderly. Improving metabolic and immune function of the elderly would improve quality of life and reduce risk for all other diseases. Tissue resident macrophages and the NLRP3 inflammasome are established drivers of inflammaging and metabolic dysfunction. Energy sensing-signaling pathways connect sterile and metabolic inflammation with cellular senescence and tissue dysfunction. We discuss recent advances in the immunometabolism field. Common themes revealed by recent publications include the alterations in metabolic signaling (SIRTUIN, AMPK or mTOR pathways) in aged immune cells, the impact of senescence on inflammaging and tissue dysfunction and the age-related changes in metabolic tissues, especially adipose tissue, as an immunological organ. Promising gerotherapeutics are candidates to broadly target nutrient and energy sensing, inflammatory and senescence pathways, and have potential to improve healthspan and treat age-related diseases.

Introduction

Age is the greatest risk factor for chronic diseases such as metabolic disease, cancer and neurodegenerative diseases [1]. The pillars of aging, including inflammation and metabolism, define key mechanistic areas of research that are critical to the aging process and age-related diseases [2]. Inflammaging, described as the accumulating chronic low-grade inflammation with age, drives age-related pathology including metabolic disease, autoimmunity and frailty [3]. In metabolic tissues, inflammaging leads to excess adiposity, impaired metabolism, impaired lipolytic signaling and reduced metabolic flexibility. Recent work has identified direct links of inflammaging and metabolic dysfunction to cellular senescence. Although further work is still needed to clarify how immunosenescence is a part of cellular senescence, this research identifies intersecting points of immunometabolism, immunosenescence and inflammaging at the molecular or cellular level and within critical tissues for a greater understanding of age-related diseases.

Cellular senescence is a cell state where irreversible cell cycle arrest is elicited via various cellular stresses or macromolecular damage [4]. Senescent cells accumulate with age, especially in metabolic tissues such as white adipose tissue (AT) and liver, and this accumulation inhibits lifespan and healthspan [5,6]. The characteristics of senescent cells include an inability to proliferate, with activation of p53/p21 or pRb/p16 pathway, and production of a high level of senescence associated secretory products (SASPs), such as IL-1β, IL-6 and MCP-1 [4,7,8]. Their accumulation can be driven by increased expression of inhibitory molecules permitting escape from immunosurveillance and suboptimal cytotoxicity from aged immune cells [9,10]. The SASPs not only mediates activation of tissue resident cells, immune cell infiltration, but also alters nicotinamide adenine dinucleotide (NAD) metabolism, a central metabolite in energy metabolism [4,11]. SASP factors stimulate expression of CD38, a NADase on the surface of cells including immune cells, which are responsible for the decline of NAD in aging [11-13]. Interestingly, cellular senescence can be modulated by differential NAD metabolic changes. High NAD⁺/NADH ratio mediated by the high mobility group A proteins (HMGA) - nicotinamide phophoribosyltransferase (NAMPT) axis increases glycolysis, mitochondrial respiration and SASP in oncogene-induced senescence [14]. In contrast, mitochondrial dysfunction-mediated senescence is partly attributed to lower NAD⁺/NADH levels and sustained activation of the NAD-AMPK signaling pathway [15]. The family of SIRTUIN proteins, NAD⁺ dependent deacetylases, are downregulated in senescent non-immune cells and aged immune cells and also have been identified to drive cellular senescence [13,16].

The SASP, SIRTUINS and NAD metabolism also provide new links to inflammaging, especially those induced by the NLRP3 inflammasome, a canonical sensor of a wide-range of damage signals (damageassociated molecular patterns: DAMPs) that accumulate with aging (Figure 1). SASP production by senescent cells is regulated by the inflammasome and IL-1 signaling; furthermore, certain SASP factors themselves may act as DAMPs [12]. The NLRP3 inflammasome, is highly expressed in tissue resident macrophages, and it’s activation by DAMPs results in NF-κB activation, NLRP3 inflammasome complex formation, and caspase 1-dependent release of IL-1β and IL-18. The role for the NLRP3 inflammasome is demonstrated in aged Nlrp3-deficient mice, which have restored glucose metabolism, catecholamine-induced lipolysis, bone density, and cognition, among other improvements in healthspan [17-20]. Aged knockout mice also show increased autophagy, NAD⁺ levels and SIRT1 protein [17]. Furthermore, small molecule inhibition of the NLRP3 inflammasome in animal models of Alzheimer’s Disease, atherosclerosis and myocardial infarction inhibit inflammation and have beneficial effects in preventing disease or preserving tissue function [21-24]. The hyperactivation of inflammasome and SASP-mediated inflammation are likely to be promoted by declining autophagy in aging [12,25]. The full extent of autophagy or other pathways as connecting cell-autonomous or non-cell-autonomous factors between inflammasome activation and senescence accumulation during aging is unknown.

Figure 1. Immunometabolism during inflammaging.

A. Sequence of events leading to inflammaging and metabolic dysfunction in tissues. B. Schematic to describe the triggers of inflammation, the responding tissue resident myeloid cells in AT and liver, the pathways that are activated within those myeloid cells or in neighboring cells, the interacting cell types and the resulting metabolic and tissue dysfunction. C. Pathways of energy or nutrient sensing and NLRP3 inflammasome activation. Alterations in energy sources, including (1) sensing of DAMPs to activate the NLRP3 inflammasome pathway or (2) changes in ATP/ADP and NAD+/NADH to drive activation of the sirtuin or AMPK pathway. Mitochondrial dysfunction that results in elevated reactive oxygen species (ROS), mitochondrial (mt) DNA or altered NAD+/NADH levels are common themes that activate and alter these pathways in aging.

Immunosenescence is a component of inflammaging and refers to the dysregulation of the aged immune system, indicating both the inappropriate dysfunctional cells and the hyperactive cells, but not the cellular senescence of immune cells. Immune cells may express commonly used senescence markers, p16^INK4a and p21^CIP1; however, it is controversial as to whether the cells are truly senescent, as these kinase inhibitors are also required for immune cell differentiation. In this review, we focus on the recently identified changes in metabolic pathways of aged immune cells to better understand immunosenescence (Figure 1&2).

Figure 2. Immunosenescence in aging.

Immunosenescence is a concept focused on the alterations in the aged immune system. Aged immune cells may be dysfunctional and inactive or hyper-inflammatory. Adaptive immunity shows increases in TNFα⁺ plasma cells producing autoantibodies and inflammatory, aged B cells (ABCs) that are unresponsive through their B cell receptor. Antigen-specific antibody production, one carbon metabolism and oxidative phosphorylation are decreased with age. There is an increased frequency of IL10⁺ regulatory T follicular helper cells that fail to interact with B cells effectively, ultimately results in reduced T cell priming. T cells also show impaired metabolism, reduced SIRT1, but increased senescent features. Innate Immunity shows increases in myelopoiesis, elevated basal NLRP3 activation, decreased phagocytosis and reduced NAD metabolism. Other innate cells, including natural killer cells, eosinophils and neutrophils are altered in number and function.

Dysregulated metabolic pathways within aged immune cells

Innate Immunity:

Hematopoietic cells are predisposed towards myelopoiesis during aging. Myeloid-derived suppressor cells, a heterogenous population of myeloid cells accumulate in the marrow in a NF-κB-dependent mechanism [26], produce IL-1β, and have greater suppressive activity on T cell proliferation in aged mouse models [27]. Their IL-1β production also inhibits B cell lymphopoiesis [27], but promotes plasma cell expansion [28]. The precise contribution of myelopoiesis and recruitment versus the proliferative capacity of tissue resident macrophages and whether each is regulated by the NLRP3 inflammasome during aging is unclear.

Aged macrophages have heightened inflammatory pathways, including the NLRP3 inflammasome activation, but reduced phagocytic ability, antigen presentation, mitochondrial dysfunction and impaired cellular metabolism [29-33] (Figure 2). Macrophages from aged Nlrp3-deficient mice have reduced inflammation, in part, mediated through increased reactive oxygen levels and expression of growth differentiation factor (GDF)-3 [29,31]. Recent work highlighted the role for the de novo production of NAD via the kynurenine pathway metabolism of tryptophan, which regulates mitochondrial function, inflammation and macrophage phenotype [33]. Declines in that cell-autonomous production of NAD in aged macrophages increases inflammation and impairs oxidative metabolism [33]. Additional research shows SIRTUIN 2 (SIRT2) inhibits inflammasome activation through deacetylation [34]. These results suggest that reduced NAD metabolism also impairs SIRT2 activity leading to the NLRP3 inflammasome activation during aging. Interestingly NAD supplementation, via nicotinamide mononucleotide, reduces reactive oxygen levels, NLRP3 inflammasome activation and bone loss, suggesting that metabolic defects in aged macrophages may be overcome [35]. Further links between the de novo NAD pathway, NAD⁺/NADH levels, the sirtuin pathway and inflammasome activation in aged macrophages remain to be explored.

Other innate immune cells, including natural killer (NK) cells and eosinophils are also altered with age (see Figure 2), but which metabolic pathways are altered within these cell types remain unclear.

Adaptive Immunity:

B cell lymphopoiesis moderately declines with age. However, increased numbers of plasma cells, with elevated levels of IL-1 and TNFα, promote myelopoiesis in aging bone marrow [28]. These changes, along with altered memory B cell subsets [36], and increased accumulation of aged B cells (ABCs), contribute to impaired aged humoral responses [37]. ABCs secrete TNFα, IL4 and IL10, and respond to TLR agonists, but not B cell receptor signaling and therefore are directly linked to the reduced lymphopoiesis and reduced responses to antigen challenge during aging [37] (Figure 2).

B cells rely on glycolytic metabolism for activation and differentiation [38]. With aging there are mild decreases in glycolysis and pronounced defects in mitochondrial energy production [39]. There are also alterations in expression of genes involved in metabolism, with decreases in expression level of sirtuin (Sirt)1, FOXO1 and carnitine palmitoyl transferase in antibody secreting cells from human individuals [39]. SIRT1 is highly expressed in resting B cells and downregulated by stimuli inducing activation-induced cytidine deaminase (AID) which is required for immunoglobulin class switch DNA recombination and somatic hypermutation [40], therefore SIRTUIN signaling may also drive reduced somatic hypermutation and class-switching. These results are described in circulating B cells, but a major finding over the last few years, is the recognition of the accumulation of tissue resident B cells, especially in the white AT and liver [41,42].

Aged T cells are characterized by reduced thymic output, oligoclonality of T cell repertoire, and accumulation of highly differentiated T cells, exhausted T cells and activated regulatory T cells, possibly leading to a higher risk of autoimmunity and infection [43]. Aging causes suboptimal induction of mitochondrial biogenesis and metabolism. Upon activation with reduced levels of metabolites in glycolysis, the pentose phosphate pathway and the TCA cycle, which impairs activation [44]. Increased mTOR signaling in aged T cells via miR-21 also entails impaired tissue homing and memory cell differentiation of T cells [45,46]. Supplementation with a TCA cycle metabolite, alpha-ketoglutarate extends lifespan and, interestingly, also induces IL-10 production in T cells [47]. IL-10, traditionally an anti-inflammatory cytokine, may play a complicated role in aging. There is an accumulation of IL-10 producing T follicular helper cells with age, which ultimately curtails T cell-dependent antibody response to vaccination [48].

During aging there is an accumulation of senescent-like T cells, highly differentiated functional memory T cells that exhibit low proliferation capacity. These cells express sestrins, stress-inducible metabolic regulators known to inhibit mTORC1 signaling. Sestrins are responsible for the low responsiveness via hyperactivation of AMPK-MAPK pathway and acquisition of NK receptor and dependent cytotoxicity [49,50]. Defective SIRT1-FOXO1 axis regulates their glycolytic capacity and granzyme B production, although the specific role of these memory T cells in aging needs to be investigated [51]. T cells with dysfunctional mitochondria by deficient mitochondrial transcription factor A (TFAM), a key regulator of mitochondria DNA, have impaired activation and induce inflammaging, as well as premature aging in mice [52]. These results demonstrate the importance of T cell-intrinsic metabolism on age-related pathology.

Association of aged tissue resident immune cells with metabolic dysfunction

Adipose tissue (AT):

AT is a critical organ in energy balance and nutritional homeostasis. Increased adiposity and metabolic dysfunction during aging is highly dependent upon the NLRP3 inflammasome-induced inflammaging [29,42,53]. White adipocytes traditionally store lipid and use discrete mechanisms for release of the stored triglyceride. Lipolysis drives the release of free fatty acids, glycerol, and proteins to be used as metabolic and signaling substrates. AT shows reduced lipolytic signaling and reduced NAD levels, driven by accumulation of CD38 [13]. Changes in AT cellular composition including the altered immune cells and the accumulating senescent burden impairs both NAD and lipid metabolism (Figure 3). The immune cell alteration and lipolytic impairment may also be linked to impaired thermogenesis and reduced responses to infection during aging.

Figure 3. Aged tissue contains inflammatory and senescent cells contributing to metabolic dysfunction.

Schematic to highlight aged tissue. Liver and adipose tissue are two critical metabolic tissues that show alterations in immune cells, metabolic signaling and senescence. (Left) White visceral adipose tissue is the first to show elevated NLRP3 inflammasome activation, accumulating B and T cells in fat-associated lymphoid clusters and elevated levels of senescent cell burden. NLRP3-expressing macrophages are found in crown-like-structures (CLS), near sympathetic nerves (SNS), and fat-associated lymphoid structures (FALCs). CLS also have eosinophils (Eo.) in close contact, which support tissue resident macrophages. Dying adipocytes in CLS provide a source of damageassociated molecular patterns that activate macrophages and drive FALC accumulation. B cells in FALCs secrete antibodies, express the IL-1 receptor (IL-1R) and respond to NLRP3-induced IL1β by proliferation. Macrophages near SNS upregulate catecholamine degradation and inflammatory genes driving impairing of lipolysis. Senescent preadipocytes (Snc. Pre-AD) secrete SASP factors and interact with immune cells in ways that are not yet clear. (Right) B cells are increased in aged liver. Aged Kupffer cells (KC) upregulate the NADase, CD38, and senescence marker p21, but have lower levels of SIRT3, contributing to impaired NAD levels in liver. Mitochondrial DNA Cytokines from immune cells and SASP factors from senescent cells are increased in aged tissues. IL1β is a SASP factor secreted from KCs and Senescent liver sinusoidal endothelial cells (Snc. LSECs) which increases lipid accumulation via PPARα and SREBP1c in hepatocytes.

Resident visceral AT F4/80⁺ CD11b⁺ SiglecF⁻ macrophages are numerically decreased, although they express an elevated inflammatory profile, with increased NLRP3 inflammasome activation that is at least partially regulated by GDF3 [29,30]. F4/80⁺ CD11b⁺ SiglecF⁺ eosinophils, critical regulators of insulin sensitivity and inflammation during obesity, are also decreased with age. Eosinophil production of IL-4 recruits alternatively activated macrophages and improves insulin sensitivity [53]. Aged-Nlrp3 deficient mice have increased (restored) AT macrophages. Along with decreased myeloid cells, there are expansions in adaptive immune system, include T and B lymphocytes [42,54]. Aged adipose B cells are a subset of the ABCs, with a few distinct differences. They express the IL-1 receptor, and their proliferative capacity requires the NLRP3 inflammasome and IL-1 signaling [29,42]. A subset of AT B cells are plasma cells, expressing IgM antibodies, and may contribute to the global increase in autoantibodies [41]. Functionally, aged murine AT macrophages upregulate catecholamine-degradation machinery, reducing lipolytic signaling in adipocytes [29]. Aged AT B cells also mediate lipolytic impairment although the precise mechanisms are unclear [42]. Overall, these data point towards a sequential series of events, likely connected by specific inflammatory signals such as senescent cell accumulation leading to metabolic dysfunction (Figure 3).

AT microenvironment niches are altered with age. Macrophages are abundant throughout the parenchyma, surrounding crown-like-structures and lining both sympathetic nerves and blood vessels, whereas eosinophils are found surrounding crown-like-structures [53]. In the aged visceral AT, but not other depots, lymphoid clusters, termed fat-associated lymphoid clusters (FALCs), which harbor the expansion of T and B lymphocytes, are increased [42,55]. Lymphoid clusters develop in response to acute or chronic inflammation and are an essential component of infection clearance [56]. Given the increase in Tfh cells and B cells during aging, but with impaired adaptive and humoral responses, it remains to be seen whether aged FALCs are immunologically responsive. Interestingly, the accumulation of lymphoid clusters does occur in other aged tissues, including the bladder [57], but direct links between metabolic signaling, senescence and FALC formation need to be dissected out (Figure 3).

Liver:

Aging reduces liver volume, disrupts blood flow and increases susceptibility to liver fibrosis, non-alcoholic fatty liver disease, and liver injury [58]. AT proximity and anatomical location to the liver, as well as its propensity for secreting various factors, makes the AT a prime suspect for cross-tissue regulation of inflammation and metabolic dysfunction in the liver with age. Similar to AT, liver shows elevated senescence impaired metabolism and an expansion of B cells [13,41]. Kupffer cells, liver resident macrophages, are important in liver homeostasis, but there is limited data regarding their roles in aging. Aging upregulates CD38 expression in Kupffer cells, resulting in NAD decline and SIRT3-dependent mitochondrial dysfunction [13,59]. The increase in CD38 expression on Kupffer cells was paralleled by increased senescent cell marker, p21^CIP1, and increased signature of pro-inflammatory M1-like cells. Cellular senescence is likely to participate in some areas of age-related liver pathology, as liver fat accumulation may be accelerated by the increase in senescent p16^INK4A+ LSEC and Kupffer cells [60] (Figure 3).

Mechanistically, the NLRP3 inflammasome and IL-1β drive the pathology of aged liver, as Nlrp3-deficiency rescued age-related glucose intolerance and high cholesterol [61]. In liver injury, aging enhances cGAS/STING-mediated NLRP3 activation by mitochondrial DNA in macrophages, worsening liver ischemia and reperfusion injury [62]. Similarly, endotoxin-induced liver inflammation is also slowly resolved during aging, resulting in chronic activation of NLRP3 and IL-1β production [63]. The increased IL-1β induces lipogenic machineries via PPARα and SREBP1c to drive lipid accumulation in aged hepatocytes, indicating a direct link between NLRP3 inflammasome and liver tissue lipid accumulation [63] (Figure 3).

Senolytic therapy as gerotherapeutics to target immunometabolism

With aging, two immunological features, inflammaging and immunosenescence are interconnected through cell-specific metabolic changes. There are many gerotherapeutics targeting the pillars of aging, aimed at improving healthspan and which have beneficial effects on AMPK-mTOR (e.g., metformin, rapamycin) and SIRTUIN signaling (e.g., resveratrol), autophagy or reducing inflammation [4]. (See Table 1 for a list of recent publications using gerotherapeutics). Based on the research revealing the interactions of these pillars of aging, gerotherapeutics that directly target a single pillar may also indirectly affect all pillars. Senolytics, agents that induce the death of senescent cells specifically are currently in clinical trials as a new type of gerotherapeutic for improving age-related diseases [4,11,64,65]. Both natural senolytics, such as fisetin, and FDA approved compounds, such as dasatinib in combination with quercetin, selectively eliminate senescent cells resulting in improved immune cell frequencies and reduced inflammation in addition to the improved health span benefits [4,64]. Senolytics may also remove p16^Ink4a+ Kupffer cells, prevent DC activation and Th1/Th17 immunogenicity by limiting mtDNA from senescent cells, which in turn improve tissue homeostasis [60,65]. In the future, additional research is required to reveal the impact of senolytics on aged immune system and the affected immune cell types to improving health span during aging.

Table 1.

Effects of gerotherapeutics on immune cells. IRI: Ischemia-reperfusion injury; S.pnm.: Streptococcus pneumoniae

Drug	Model	Effects	Reference
Rapamycin	• C57BL/6 mice • 22–24m • S. pnm.	• Conferred modest protection against mortality. • Diminished lung damage rather than reduced bacterial burden. • No effect on levels of SASPs in whole lung homogenates.	[66]
	• C57BL/6 mice • 16–18m • West Nile virus	• Reduced thymic cellularity. • Reduced the mortality, but not significant.	[67]
mTOR inhibitor (RAD001, BEZ235)	• Human subjects • ≥ 65 years • Influenza vaccine	• Induced higher antibody titers. • Improved protection against influenza infection.	[68]
Dasatinib+Quercetin (D+Q)	• C57BL/6 mice • 18m • IRI	• Reduced systemic levels of CD8+ IFN-γ+, CD4+IFN-γ+, and CD4+IL-17+ cells after renal IRI.	[65]
	• p16-Cre/R26-mTmG mice • 10m	• Removed p16High F4/80+ cells in liver.	[69]
Metformin	• Human PMBC • Avg 62 years	• Ameliorated the Th17 profile by increasing autophagy and improving mitochondrial bioenergetics.	[70]