Metabolic Alterations in Aging Macrophages: Ingredients for Inflammaging?

TREIMM 1536 No. of Pages 15

Opinion

Metabolic Alterations in Aging Macrophages: Ingredients for Inflammaging? Adriaan A. van Beek,1,2,3 Jan Van den Bossche,4,5 Pier G. Mastroberardino,6 Menno P.J. de Winther,5,7 and Pieter J.M. Leenen3,* Aging is a complex process with an impact on essentially all organs. Declined cellular repair causes increased damage at genomic and proteomic levels upon aging. This can lead to systemic changes in metabolism and pro-inflammatory cytokine production, resulting in low-grade inflammation, or ‘inflammaging’. Tissue macrophages, gatekeepers of parenchymal homeostasis and integrity, are prime inflammatory cytokine producers, as well as initiators and regulators of inflammation. In this opinion piece, we summarize intrinsic alterations in macrophage phenotype and function with age. We propose that alternatively activated macrophages (M2-like), which are yet pro-inflammatory, can accumulate in tissues and promote inflammaging. Age-related increases in endoplasmic reticulum stress and mitochondrial dysfunction might be cell-intrinsic forces driving this unusual phenotype. Macrophages and Inflammaging Systemic decline during aging is characterized by various changes at the cellular level, summarized in a landmark review [1]. These features comprise genomic instability, epigenetic (see Glossary) changes, increased protein misfolding, mitochondrial dysfunction, and dysregulated nutrient sensing. Moreover, the ability to restore homeostasis via proteasomal degradation, the unfolded protein response (UPR) and autophagy decrease with aging, leading to fragile conditions in which cells lose proper function and either die or enter a senescent state [2,3]. This can be accompanied by systemically increased pro-inflammatory factors such as IL-1, IL-6, IL-8, TNF, and C-reactive protein [4]. While these mediators serve a homeostatic role in acute inflammation, their chronic elevation has been associated with diseases such as diabetes, atherosclerosis, or autoimmunity. In some cases, senescent tissue cells can secrete a variety of inflammatory cytokines and chemokines, a phenomenon described as the senescence-associated secretory phenotype (SASP) [5]. It is undecided whether these SASP-positive parenchymal cells are the major cellular sources of ‘inflammaging’ mediators, or whether activated immune cells are critical contributors to the increasing concentrations of such mediators in the steady state during aging. Macrophages are critical regulators of processes aimed at maintaining homeostasis and prominently contribute to inflammatory and immune responses, but also help maintain metabolic stability [6]. Direct evidence on the role of macrophages in determining life span and inflammaging is scarce. A study in Drosophila showed a link between reduction in life span by a lipid-rich diet and increase of the macrophage-derived cytokine encoded by upd3 [7]. Silencing of upd3 in macrophages rescued insulin sensitivity and life span [7]. In addition, long-term selective elimination of macrophages in aging mice has been shown to inhibit peripheral nerve degeneration and reduction in muscle strength [8]. Moreover, macrophage depletion in aged mice can prevent strongly increased pro-inflammatory cytokine concentrations and deaths of

Trends in Immunology, Month Year, Vol. xx, No. yy

Highlights Aging is associated at the cellular level with several adaptations, fueled by increasing damage and reduced capacity for repair. This generates a condition of low-grade inflammation, called ‘inflammaging’. Macrophages are prime cells in initiation and regulation of inflammatory processes and may thus play major roles in inflammaging. Macrophage polarization and activation, induced by intrinsic or extrinsic conditions, are reflected in and regulated by the cells’ metabolic and epigenetic profiles. Age-induced changes in macrophages are diverse and, in general, may represent pro-inflammatory activation of cells with an alternatively activated (M2-like) phenotype.

1 Top Institute Food and Nutrition, Nieuwe Kanaal 9A, 6709 PA Wageningen, The Netherlands 2 Cell Biology and Immunology Group, Wageningen University, De Elst 1, 6709 PG Wageningen, The Netherlands 3 Department of Immunology, Erasmus University Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands 4 Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Molecular Cell Biology and Immunology, Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam, De Boelelaan 1117, Amsterdam, Netherlands 5 Amsterdam UMC, University of Amsterdam, Experimental Vascular Biology, Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences,

https://doi.org/10.1016/j.it.2018.12.007 © 2018 Elsevier Ltd. All rights reserved.

1

TREIMM 1536 No. of Pages 15

animals upon systemic immune stimulation (high-dose anti-CD40/IL-2 or IL-2/IL-12), compared with young mice that survive the same treatment [9]. Macrophages are indeed thought to be central to the inflammaging process [10]; they are extremely versatile, responding to environmental triggers and adapting their phenotype and function accordingly (Box 1). In this opinion article, we integrate current knowledge on inflammatory aspects of macrophage activation related to the aging process. We discuss to what extent aging-related intrinsic changes in macrophages and their bone marrow precursors contribute to the systemic condition of inflammaging. We posit that extrinsic factors derived from the aging cells’ environment, such as increased parenchymal cell debris and changes in stromal functions, may drive macrophage activation. These extrinsic factors have been recently reviewed in the context of the inflammaging process [11]. Macrophage-intrinsic age-related changes are triggered by increased damage at the protein, lipid, and nucleic acid levels, leading to senescence and organelle dysfunction [2]. Recent findings, presented in this article, have highlighted the close connections that exist between aging, macrophage metabolism, epigenetic status, and inflammatory states. Insight into this matter may reveal putative targets for therapeutic intervention in the inflammaging process in a variety of conditions and will ideally provide directions for future research.

Phenotypic and Functional Changes in Aging Macrophages Exposure to altered factors in an aging cellular environment and intrinsic changes in aging macrophages are both likely to play a role in phenotypic and functional changes in macrophages (see below). Table 1 summarizes data on age-related changes in human and mouse macrophages (see also supplemental Table S1 online). Of note, most reported age-related alterations in macrophage functions are based on ex vivo measurements, which reflect the cells’ functional potentials upon challenge, rather than their steady state in vivo activity. It is evident that major differences are found in pro-inflammatory cytokine production depending on the tissue of origin, as concluded in comparative studies [12]. Indeed, the local microenvironment plays a critical role in shaping the macrophage epigenetic landscape and gene expression. For instance, mouse alveolar macrophages uniquely express Car4, and peritoneal macrophages Tgfb2 [13]. These tissue-specific expression patterns have been further confirmed by transferring peritoneal macrophages to the lung, where reprogramming occurred, including the upregulation of Car4 and downregulation of Tgfb2. Despite differences between distinct populations, some general messages about age-related changes in macrophage phenotype and function may be derived. Macrophage numbers

Box 1. Macrophage Polarization: Beyond Pro-inflammatory M1 and Anti-inflammatory M2 As sentinel cells, macrophages are able to respond swiftly to changes in their environment, such as alterations in cytokine milieu, microbial ligands, and immune complexes, and adapt their phenotype and function accordingly. Initially, their state of polarization was defined binarily as classically (M1) or alternatively activated (M2). However, it is now realized that these stages represent ends of a spectrum in a multidimensional space [83,84]. In general, classically activated M1 macrophages are catabolic, pro-inflammatory cells involved in antimicrobial host defense, while M2 macrophages are anabolic cells frequently counteracting inflammation and stimulating tissue repair. This concept, however, is not written in stone, since M2-polarized macrophages may also produce significant amounts of pro-inflammatory cytokines such as TNF, IL-1, and IL-6 upon stimulation with immune complexes and TLR ligands [85] or experimental manipulation affecting their autophagy capacity [42]. For ease of communication, we indicate macrophage stages as M1- or M2-like but it should be realized that this is an oversimplification, and that M1- and M2like polarization are not synonymous with pro- and anti-inflammatory activity, respectively, as is often stated. For instance, macrophages seem to become more pro-inflammatory, yet M2-like, with aging [28–31].

2

Trends in Immunology, Month Year, Vol. xx, No. yy

Meibergdreef 9, Amsterdam, The Netherlands 6 Department of Genetics, Erasmus University Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands 7 Institute for Cardiovascular Prevention (IPEK), Munich, Germany

*Correspondence: [email protected] (Pieter J.M. Leenen).

TREIMM 1536 No. of Pages 15

Table 1. Age-Dependenta Alterations in Human and Mouse Macrophages Tissue Mfb

Numbers

BMDM MDMd Lung

=/"

Peritoneum

=/"

Spleen

=

WAT

=

Skin

=d

Muscle

"

Brain

"

Glossary

Phagocytosis

LPS responsec

#/=

" "d "

#/=

# # "

#

#

"

Increase ("), equal (=), or decrease (#) as compared with young macrophages. Additional information, such as response to other TLR ligands or microbes and references to individual studies, is provided in supplemental Table S1 online. BMDM, monocyte-derived macrophages. a Young (human: 30 years old; mouse: 6 months old) versus old (human: 65 years old; mouse: 18 months old). b Tissue macrophages concern mouse cells, unless indicated as human macrophages. c Secretion of pro-inflammatory cytokines (IL-1b, IL-6, TNF) in response to LPS. d Human macrophages.

remain stable in mouse skin, spleen, and white adipose tissue (WAT) (e.g., [14]), and increase in mouse muscle and brain and human muscle (e.g., [15]). Their phagocytic capacity can decrease with aging or remains unchanged; for instance, mouse peritoneal macrophages and microglia may show an age-related decline in phagocytosis, but this trait has been found unaltered in macrophages derived in vitro from bone marrow precursors [bone marrow-derived macrophages (BMDM)] in one study [16] but not in another [17]. Production of inflammatory mediators upon Toll-like receptor (TLR) triggering has been reported to be increased upon aging in most studies, as exemplified by enhanced TNF and IL-6 production in human and mouse precursor-derived macrophages in response to LPS [9]. This increased inflammatory set-point has been associated with systemically increased expression and activation of components of the NLRP3 inflammasome in mouse myeloid cells [18]. In addition, a recent study suggested that aged macrophages were inhibited from terminating TLR responses relative to controls [19]. Specifically, LPS stimulation of peritoneal macrophages from aged mice resulted in similar Tnf transcript levels as their young counterparts. TLR4 signaling blockade after 10 minutes of LPS stimulation led to prolonged high concentrations of Tnf, Il-1b, and Il-6 transcripts in aged relative to young macrophages [19]. However, conclusions on age-dependent alterations in TLR responses of macrophages are not uniform and reports have also shown diminished pro-inflammatory cytokine responses to TLR2 and TLR4 ligands in aged splenic or alveolar macrophages relative to young ones (e.g., [20,21]). This discrepancy may be partially explained by the different experimental settings (possibly including TLR ligands, microorganisms, different ontogenetic origins of responding cells) [22], but further investigation is thus warranted. Production of the anti-inflammatory cytokine IL-10 is enhanced with aging upon stimulation of mouse splenic macrophages and BMDM (e.g., [23,24]). Also, the production of prostaglandin (PG)E2, which contributes to age-associated suppression of T cell function, is generally increased in murine peritoneal macrophages with aging, related to increased cyclooxygenase 2 (COX2) activity in aged macrophages compared with young macrophages [25,26]. A recent study noted significant in vivo accumulation of macrophages with high expression of p16INK4a and b-galactosidase upon aging of p16Ink4a/LUC mice. Both markers have

Advanced glycation endproducts: proteins that have been modified by nonenzymatic covalent binding of glycans such as glucose; formed in a multistep process. Autophagy: cellular process to recycle dysfunctional or unnecessary cell material, also used during starvation to yield nutrients. Berberine: drug that activates AMPK via inhibition of mitochondrial respiratory chain complex I. Possesses antidiabetic, antimicrobial, and antidiarrheal activity. Endoplasmic reticulum (ER) stress: cell stress induced by accumulation of unfolded or misfolded proteins in the ER, typically related to high demand of protein synthesis in the ER. Epigenetics: chromatin structure, affected by DNA (de)methylation, histone (de)methylation, and histone (de)acetylation, regulates gene accessibility and subsequent enhanced/diminished transcription. Inflammaging: chronic low-grade inflammation that occurs with aging and is characterized by dysregulated inflammatory responses, in general resulting in increased autoinflammatory responses and diminished responses to pathogens like influenza. M1 macrophages: classically activated macrophages that are induced by exposure of unpolarized macrophages to stimuli such as LPS and IFN-g. These cells express iNOS and produce nitric oxide, reactive oxygen species, as well as proinflammatory cytokines. Their main function is in host defense against microbial pathogens. M2 macrophages: alternatively activated macrophages that are induced by exposure of unpolarized macrophages to various stimuli, including IL-4, IL-10, or glucocorticoids. These cells express arginase and synthesize polyamines. Their main function is in tissue repair. The general concept of M1 versus M2 polarization, however, is now considered an oversimplification. Macrophage activation: induction or increase in one or more functions by a specific stimulus, typically reflected in the cells’ phenotype. Frequently, macrophage activation is used synonymously with

Trends in Immunology, Month Year, Vol. xx, No. yy

3

TREIMM 1536 No. of Pages 15

been associated with a senescent state when expressed in parenchymal cells, as demonstrated by their increased accumulation with age and with SASP [27]. Expression of both markers was induced in mouse peritoneal macrophages upon transplantation with senescent cells, and associated with a designated M2-like polarization phenotype [28] (Box 1). This increase in M2 polarization of tissue macrophages in steady state upon aging is supported by early observations of increased basal expression of transglutaminase-2, an M2 marker conserved with age in mouse and human [29,30]. Transglutaminase-2 activity is suggested to contribute significantly to changes in extracellular matrix in aging connective tissue [31]. Thus, M2-like macrophages producing inflammatory mediators seem to accumulate with age. In sum, macrophages can be increasingly dysregulated with aging, affecting their functions in host defense and inflammation. The observed changes, however, are not uniform, and reflect the heterogeneity of the cell type. Indeed, we argue that putative contributions of changing environmental conditions and intrinsic alterations to aging can alter macrophage phenotypes and functions.

Potential Extrinsic versus Intrinsic Causes of Macrophage Changes with Aging Alterations in exogenous stimuli probably contribute significantly to the inflammatory profile of macrophages with aging. For instance, a decrease in gut barrier function, as indicated by reduced transepithelial electric resistance, has been proposed as a major cause of increased concentrations of TLR ligands in circulation in humans with aging [32]. Also, other pro-inflammatory triggers, such as advanced glycation endproducts and S100A8/A9, can increase systemically with age in various tissues in humans and mice, activating macrophages via RAGE and TLR4 [11]. Moreover, concentrations of antiinflammatory dehydroepiandrosterone (DHEA) decrease in aging humans [33]. Decreasing vascular function with age can gives rise to increased tissue hypoxia, known to be a strong activator of macrophages [34]. This appears to be mediated via HIF-1a protein stabilization, simulating and cooperating with NF-kB activation, and leading to pro-inflammatory cytokine and reactive oxygen species (ROS) production, as shown in human and mouse cell lines. Macrophage activation by this means, however, is not easily interpreted in terms of classic M1-like versus alternative M2-like polarization based on cellular immunophenotype [34]. Together, these findings contribute to the view that the aged microenvironment can provide increasing levels of pro-inflammatory triggers that might majorly impact macrophage functions, although direct demonstration of functional alterations at the tissue level with age are clearly warranted. In addition to extrinsic influences, age-associated intrinsic cellular changes may also impact macrophage function. This is exemplified by the finding that BMDM generated from young or aged mice still show certain phenotypic and functional differences, despite extensive in vitro expansion of bone marrow precursors [9,18,24,35] (Table 1). Using mass spectrometry analysis of intracellular metabolites, one study reported that aged mouse BMDM showed a significantly blunted metabolic switch towards glycolysis and delayed increase in arginine metabolism upon LPS stimulation relative to young macrophages [36]. Lastly, we would argue that resident tissue macrophages, like parenchymal cells, can accumulate cellular damage and related adaptational changes upon aging, since these cells are generally long-lived [37]. Therefore, we survey parameters of generic cellular alterations that can be observed in macrophages upon aging: these include endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and disturbed cellular metabolism. 4

Trends in Immunology, Month Year, Vol. xx, No. yy

macrophage polarization, and indicated as classically or M1activated versus alternatively or M2activated (see below). We propose to separate activation (reflecting the magnitude of the cellular response and quantitative difference from steady state) from macrophage polarization. Macrophage polarization: the direction of the functional and phenotypic response induced by a specific stimulus. We propose to separate polarization (reflecting the direction of the response and qualitative difference from the unpolarized steady state) from macrophage activation. Metformin: a drug that decreases glucose production by the liver and increases insulin sensitivity. First-line treatment in diabetes type 2 patients. NLRP3 inflammasome: cytosolic protein complex that, when assembled upon triggering, activates caspase-1. This subsequently can cleave pro-IL-1b and pro-IL-18 to generate mature IL-1b and IL-18. Phenformin: a drug that activates the UPR via AMPK activation. Antidiabetic drug, but removed from the market due to its liver toxicity. Resveratrol: polyphenol compound with antitumor, antioxidant, and phyto-estrogenic activities. S100A8/A9: a heterodimeric Ca2+binding protein complex, (calprotectin or MRP8/14). It is present in the cytosol of multiple cell types, particularly abundant in neutrophils and recent bone marrowemigrant monocytes. S100A8/A9 molecules are released in circulation and concentrations are associated with systemic inflammation. Senescence-associated secretory phenotype (SASP): cells that enter a state of senescence, in which cellular replication is inhibited, remain metabolically active and may develop a pro-inflammatory phenotype characterized by secretion of cytokines such as IL-6 and CXCL8, proteases, and other factors like PGE2 and fibronectin. TLR ligands: ligands such as LPS or dsRNA that trigger signaling of Toll-like receptors (TLR). This interaction typically induces proinflammatory responses in stimulated cells. TLR ligands are mostly of microbial origin, although

TREIMM 1536 No. of Pages 15

ER Stress, Mitochondrial Dysfunction, Autophagy, and Inflammation We propose that changes in distinct interconnected, aging-related cellular processes might contribute to the explanation of ensuing M2-like polarization and pro-inflammatory activation of macrophages, namely associated with increased ER stress, decreased proteasomal and autophagic waste removal, and mitochondrial dysfunction. Circumstances that cause overload of misfolded or unfolded proteins in the ER, such as nutrient excess or infection, cause ER stress and activation of the UPR (Box 2). It has been shown in several mammalian species and cell types that aging is associated with reduced proteasomal activity and decreased UPR capacity to resolve ER stress [38]. Autophagy capabilities can also decrease with advanced age, as shown in various cell types, including mouse BMDM [35]. When ER stress remains elevated compared with homeostatic conditions in young individuals, this can operate in synergy with TLR signaling in mouse and human macrophages to stimulate pro-inflammatory cytokine expression [39,40]. In accordance, several studies have shown that reduced autophagy potential, as modeled in Atg5- or Atg7-deficient mouse macrophages, is related to increased production of pro-inflammatory cytokines, in particular IL-1 and IL-6 (e.g., [41,42]).

endogenous molecules can also trigger TLR. Trained innate immunity: this term is used to indicate enhanced responses by innate immune cells, such as monocytes, macrophages, and NK cells, upon repeated exposure to an activating trigger, mostly of microbial origin. This ‘innate memory’ has an epigenetic basis, which mediates changes in cellular metabolism. Unfolded protein response (UPR): this cellular response is activated upon accumulation of unfolded or misfolded proteins in the endoplasmic reticulum. It results in pause of translation, degradation of improperly folded proteins, and increased generation of molecular chaperones (Box 2).

If not due to intrinsic causes, then ER stress in neighboring parenchymal cells might be transferred to tissue macrophages, as indicated by the upregulated expression of UPR components such as grp78 and CHOP [43,44]. These effects can be mediated by soluble factors such as interleukins IL-4, IL-10 and by apoptotic bodies produced by the stressed cells that stimulate expression of ER stress markers by neighboring macrophages [44]. A remarkable functional interaction exists between ER stress, autophagy, and macrophage polarization and activation, since ER stress induces, and appears to be required to generate an M2-like phenotype in mouse and human macrophages, as evidenced from the expression of specific markers, including arginase-1 [45,46]. In agreement, transferred ER stress can also skew towards M2 polarization, as evidenced by cytokine and marker expression in human macrophages in vitro [44]. Common denominators between ER stress, autophagy, mitochondrial dysfunction, and inflammatory activation are perturbed homeostasis of cellular calcium and oxidative stress [47]

Box 2. ER Stress and the UPR in Aging The endoplasmic reticulum (ER) contributes to the formation of autophagosomes and is important in the proper folding of secreted proteins. A high demand for synthesis of secretory proteins is a source of stress for the ER. ER stress induces the unfolded protein response (UPR) by activating inositol-requiring protein 1a (IRE1a), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6) (Figure I). To restore equilibrium in the ER, information about the folding status of proteins in the ER is transduced by these three stress sensors to the cytosol and nucleus. Chronic ER stress can lead to apoptosis of damaged cells [86]. Of note, the UPR is involved in many processes, such as glucose and lipid metabolism and cell differentiation, and drives inflammation via multiple routes [87]. X box-binding protein 1 (XBP1), downstream of IRE1a, inhibits the transcription factor forkhead box O1 (FOXO1), which is crucial in glucose homeostasis [1] (see also Box 3). The UPR activates macroautophagy [88]. The nutrient sensor mTORC1 selectively suppresses IRE1a activation [89]. Elevated ER stress drives M2-like macrophage polarization in a c-Jun N terminal kinase (JNK)-dependent manner [45], and contributes to inflammation of adipose tissue with aging [90]. Aged adipose tissue macrophages show increased ER stress, accompanied by increased TNF production [90]. TLR agonists work synergistically with ER stress in the production of pro-inflammatory cytokines [39,40]. With aging, many components of the UPR show reduced expression and activity [38]. Moreover, ER stress is implicated in metabolic and age-related diseases, such as diabetes, atherosclerosis, Alzheimer’s, and Parkinson’s disease [38].

Trends in Immunology, Month Year, Vol. xx, No. yy

5

TREIMM 1536 No. of Pages 15

Taken together, ER stress, the UPR, and mitochondrial dysfunction play a role in key processes that are altered significantly with aging, such as glucose metabolism, autophagy, and pro-inflammatory cytokine production. These processes are also closely involved with macrophage activation and polarization.

ER stress

mTORC1

IRE1

PERK

ATF6

Inflammaging

UPR

XBP1 Autophagy FOXO1

Figure I. Endoplasmic Reticulum Stress and the Unfolded Protein Response in Aging. ATF6, Activating transcription factor 6; ER, endoplasmic reticulum; FOXO1, forkhead box O1; IRE1a, inositol-requiring protein 1a; PERK, protein kinase RNA-like ER kinase; UPR, unfolded protein response; XBP1, X box-binding protein 1.

(Figure 1A, Key Figure). Mitochondria take up Ca2+ released from the ER at the ER–mitochondrial contact sites and function as buffers of cytosolic Ca2+ levels. Mitochondrial dysfunction, as occurring with aging and upon M1-polarization, can decrease membrane potential and therewith Ca2+ uptake potential, leading to increased cytosolic Ca2+ [47]. This mediates activation of several pathways, including NFAT, MAP kinases, and the NLRP3 inflammasome [48,49]. Consequently, inflammatory parameters can be increased, as indicated by nuclear translocation of NF-kB and secretion of IL-1b. Moreover, increased production of ROS, associated with mitochondrial dysfunction, can stimulate inflammasome activation and IL1b release [50]. Thus, we consider increased cytosolic Ca2+ concentrations and oxidative stress important drivers of age-related inflammation in macrophages. Taken together, it can be envisaged that, at the macrophage level, increased ER stress and oxidative stress with aging can contribute to enhanced production of pro-inflammatory cytokines, and thus to systemic inflammaging. However, since ER stress can stimulate M2 polarization, this may lead to an unusual M2-like pro-inflammatory profile. It is likely that environmental factors further contribute to this phenotype, although this remains to be demonstrated.

Deregulated Nutrient Sensing and Mitochondrial Dysfunction in Macrophage-Mediated Inflammaging It is increasingly recognized that polarization and activation of macrophages is tightly linked to their energy metabolism (Box 3) [51–53]. Figure 1 summarizes multiple involved metabolic 6

Trends in Immunology, Month Year, Vol. xx, No. yy

TREIMM 1536 No. of Pages 15

Key Figure

Cellular Metabolic Processes Are Crucial in Macrophage Inflammatory Activity and Polarization, and Are Associated with the Inflammaging Process (A)

(B)

Pro-inflammatory cytokines

NF-κB

LPS IL-1β

NLRP3 inflammasome acƟvaƟon

ROS

Insulin IGF-1

PGC-1

Glucose

AP-1

ER stress (M2) [Ca2+]e

IL -4

Mitochondrial dysfuncƟon

(M1)

mTOR

p62 / SQSTM1

[Ca2+]i

Amino acids

Oxphos (M2)

Glycolysis

ROS

Autophagy

AKT

AMPK

FaƩy acids

LPS

mTOR

HIF -1α

FOXO

ROS succinate

Nrf2

SIRT1 IL-10

Mitochondrial dysfuncƟon

IL-1β

NF-κB

TLR4

IL-6 TNF

LPS

Figure 1. (A) With aging, endoplasmic reticulum (ER) stress increases due to misfolded proteins, decreased unfolded protein response capacity, decreased autophagy, and mitochondrial dysfunction. Mitochondrial dysfunction leads to reactive oxygen species (ROS) production, which in turn activates NLRP3 inflammasomes, leading to mature IL-1b protein. Cytosolic calcium ([Ca2+]i), after release from the ER, is normally taken up by mitochondria. Mitochondrial dysfunction leads to increased cytosolic calcium levels that also activate inflammasomes. ER stress activates transcription factors NF-kB and activator protein 1 (AP-1), leading to production of pro-inflammatory cytokines such as IL-6 and TNF. ER stress and mitochondrial dysfunction activate autophagy. Expression of p62/SQSTM1, a central mediator of autophagy and cytosolic protein degradation, decreases with aging, as does autophagy capacity, resulting in enhanced ER stress and mitochondrial dysfunction. Autophagy is further inhibited by mammalian target of rapamycin (mTOR), the central nutrient sensor. (B) Activation of macrophages [e.g., by LPS or hypoxia-inducible factor 1 alpha (HIF-1a)] enhances glycolysis and ROS production. HIF-1a is activated by mitochondrial dysfunction, and conversely induces mitochondrial dysfunction and IL-1b production, feeding pro-inflammatory cytokine production via NF-kB. Alternatively activated, M2-polarized macrophages mainly depend on oxidative phosphorylation (oxphos), which is induced by IL-4-activated peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1). AMPactivated protein kinase (AMPK) and sirtuin 1 (SIRT1) activate PGC-1, driving M2 polarization, while simultaneously promoting mitochondrial function. AMPK and SIRT1 also inhibit HIF-1a and NF-kB, thereby inhibiting M1 polarization, while activating PGC-1 and forkhead box proteins O1/3 (FOXO1/3). FOXO1/3 enhances IL-10 production and supports Toll-like receptor 4 (TLR4) signaling. Insulin and insulin-like growth factor 1 (IGF-1), the activities of which decline with aging, activate mTOR via AKT, while inhibiting FOXO1/3. Brown arrows indicate changes observed in levels and/or function of the respective component during aging.

pathways and indicates general up- or downregulations observed in macrophages upon aging. Distinct hallmarks of aging, including decreased autophagy, deregulated nutrient sensing, and mitochondrial dysfunction can increase glycolysis and suppress oxidative phosphorylation (oxphos). The somatotrophic axis [i.e., growth hormone: insulin-like growth factor-1 (IGF-1)/ insulin signaling (IIS)] is the central pathway in nutrient signaling and its activity generally declines with aging, as observed in a wide variety of eukaryotic cell types [1]. This might not apply unequivocally to macrophages, however, since BMDM from aged mice show increased growth hormone receptor (GH-R) expression and GH-R-dependent induction of inflammatory Trends in Immunology, Month Year, Vol. xx, No. yy

7

TREIMM 1536 No. of Pages 15

Box 3. Macrophage Polarization, Cellular Metabolism, and Inflammation Recent findings show that macrophage energy metabolism, polarization, and inflammatory function are tightly linked [51–53]. Classical (M1) activation with microbial stimuli and IFN-g enhances glycolysis, thereby fueling the macrophages with fast energy and biosynthetic precursors for the rapid killing of microbes and driving their inflammatory response. Accordingly, overexpressing the glucose transporter Glut1 in mouse macrophages stimulates a pro-inflammatory phenotype [91], whilst inhibition of glycolysis with 2-deoxyglucose blunts pro-inflammatory signaling, which is mediated via HIF-1a [92]. Conversely, M2-polarized cells primarily utilize mitochondrial oxidative phosphorylation (oxphos) as an ATP source, and its inhibition with oligomycin attenuates IL-4-induced anti-inflammatory macrophage responses [57,62]. While oxphos was originally shown to be fueled primarily by fatty acid oxidation (FAO), supporting antiinflammatory macrophage responses [62], recent studies indicate that glucose can also drive oxphos and M2 macrophage responses [57,93]. In agreement, blocking glycolysis with 2-deoxyglucose inhibits the expression of IL-4-induced genes and surface markers [57,93–95]. The necessity of FAO in IL-4-induced macrophage responses is less clear. While the FAO inhibitor etomoxir was initially demonstrated to block IL-4 responses in mouse macrophages [62], it had no effect on IL-4-induced activation of mouse and human macrophages in follow-up studies [57,96]. Also, genetic disruption of FAO did not affect IL-4-induced polarization and therefore etomoxir has been suggested to have off-target effects that are independent of FAO, as reviewed in detail elsewhere [96,97]. In the nutrient-sensing insulin/insulin-like growth factor (IGF)-1 signaling (IIS) pathway, the FOXO transcription factor family is a downstream target, which is inhibited by IIS signaling [1]. FOXO1 has been shown to stimulate both proinflammatory TLR4 signaling and IL-1b production in mouse macrophages [98], as well as the expression of the antiinflammatory cytokine IL-10 in M2 cells [99]. FOXO activity is known to extend life span in worms and flies [1], and could support the phenotypic development of aging macrophages in distinct directions. We posit that macrophage metabolism is intertwined with macrophage polarization and inflammatory activation. Targeting specific metabolic pathways in macrophages to induce repolarization and thereby modulate inflammatory responses warrants further study in the context of aging.

components [18]. Ablation of the IGF-1 receptor in mouse macrophages can lower NLRP3 inflammasome activation, and thus IL-1 production. Similarly, myeloid cell-specific insulin/IGF1 receptor deficiency can dampen inflammation elicited in mouse skin by irritants or irradiation, or, in adipose tissue by administration of a high-fat diet in mice [54,55]. Using a mouse model of NLRP3 deficiency, NLRP3 activation was shown to be important in mediating age-related inflammaging and dysfunction of multiple organs, including adipose tissue, brain, and thymus [56]. It is reasonable to speculate that macrophages might play a major role in this, but this remains to be demonstrated. From another angle, the systemic decrease in function of IIS or downstream regulators, such as AKT and mTOR, has been reported to extend organismal longevity [1]. Therefore, downregulation of IIS signaling with aging, as observed in most cell types, might serve as a protective mechanism for reducing inflammation, although this warrants further investigation. A reduction of mitochondrial oxphos with aging likely promotes an inflammatory environment, considering that mitochondrial dysfunction can prevent the reprogramming of inflammatory macrophages into anti-inflammatory cells, as evidenced from phenotypic markers and cell metabolic analysis [57]. Several molecules, including AMPK, SIRT1, and PGC-1, whose activation has been associated with increased longevity and healthy aging [1], can promote maintenance of mitochondrial function and at the same time potentiate the M2 phenotype in mouse macrophages [58]. AMPK activates sirtuins SIRT1 and 3, and vice versa. Subsequently, NAD+-dependent SIRT1 can deacetylate and inactivate NF-kB p65, thus blocking NFkB-mediated inflammation, as shown in human epithelial cells in vitro [59]. Recently, aging has been associated with decreased NAD+ synthesis in human and mouse macrophages in vitro and in vivo, as well as with a switch towards a pro-inflammatory phenotype [60]. This might provide a likely explanation for decreasing SIRT transcriptional activity in aging macrophages; SIRT1 can activate PGC-1b and thereby promote a switch from glycolysis to mitochondrial fatty acid oxidation (FAO) in human cells [61]. In agreement, the M2-polarizing cytokine IL-4 can 8

Trends in Immunology, Month Year, Vol. xx, No. yy

TREIMM 1536 No. of Pages 15

promote PGC-1b function, resulting in mitochondrial biogenesis, FAO, and oxphos in mouse macrophages [62]. We hypothesize that macrophage metabolism, polarization, and inflammatory activation are closely intertwined, and could reveal more nuances beyond the dichotomy between proinflammatory, glycolytic M1 macrophages, and anti-inflammatory M2 macrophages that utilize oxidative phosphorylation as their main source of energy. Aging is associated with shifts in nutrient sensing and metabolic pathways. Indeed, mitochondrial dysfunction and related oxidative stress in particular, together with a decreased capability to counterbalance these states, might potentially contribute to pro-inflammatory activation in aging. Despite significant recent progress in the immunometabolism field, the molecular mechanisms by which metabolic changes affect macrophage function remain largely unexplored. Various intermediates of energy metabolism can affect histone-modifying enzymes and therefore epigenetic mechanisms might contribute to translating altered energy metabolism into distinct macrophage phenotypes [63]. For example, an enhanced cytokine response of human monocytes to repeated exposure to Candida-derived b-glucan in vitro (termed ‘trained innate immunity’) was found to be associated with the induction of enhanced aerobic glycolysis governed by a specific epigenetic signature [64]. This mechanism might contribute to altering responses of macrophages to extrinsic and intrinsic triggers upon aging, as discussed below. Epigenetic regulation of macrophage activation, in the context of aging, is further discussed in Box 4.

Targets to Modulate Inflammaging The mechanisms outlined above may provide multiple access points to reduce inflammaging, either in general or targeted at macrophages (summarized in Figure 2). Dietary restriction (DR) is well known for its life span- and health span-extending effects [1,65]. These are mediated, in part, by suppressing the mTOR and IIS pathways, leading to SIRT1 activation and enhanced autophagy. DR can diminish age-dependent increases in serum IL-6 and TNF. However, after 40% DR for 20 weeks, macrophages from 6-month-old mice showed amplified pro-inflammatory cytokine production in a peritonitis model relative to macrophages from ad libitum-fed mice, correlating with reduced survival [66]. The same level of DR for 3 weeks led to better survival of 12-month-old mice after abdominal polymicrobial sepsis or endotoxemia, associated with reduced IL-6 production and WAT macrophage numbers relative to ad libitum-fed mice [67]. DR can also increase adiponectin secretion, which reduces TNF expression and stimulates polarization towards an anti-inflammatory macrophage phenotype [68]. Furthermore, lifelong 40% DR, but not lifelong exercise or ad libitum feeding, in 24-month-old mice reduced the number and activation of microglia to the level observed in 6-month-old mice [69]. These findings suggest that DR can prevent inflammaging, overall, and in macrophages. The data also underline the need for future studies addressing the questions of how long and to what extent might DR extend life, while at the same time properly maintaining macrophage innate immune functions. DR mimetics, reaching similar effects as DR without restricting energy intake, have been extensively studied. Metformin (extending mouse life span), phenformin, or berberine can activate AMPK [58]. Resveratrol mimics DR in some ways, activating AMPK and SIRT1 (in turn activating PGC-1a) [1], and blocking LPS-mediated inhibition of SIRT1 and activation of HIF-1a [70]. Rapamycin, which acts via inhibition of nutrient sensor mTOR, can also extend life span and induce autophagy in mice [1,71]. However, rapamycin may simultaneously skew macrophage polarization in vitro into pro-inflammatory IFN-g/LPS-induced M1 direction, while inducing apoptosis in IL-4-stimulated M2 cells [72]. This may contribute to unwanted side effects of rapamycin treatment, such as impaired wound healing and insulin resistance. Trends in Immunology, Month Year, Vol. xx, No. yy

9

TREIMM 1536 No. of Pages 15

Box 4. Epigenetic Regulation of Macrophage Activation in Aging Epigenetic processes, such as DNA (de)methylation and histone (de)methylation and (de)acetylation, modify chromatin structure and thus play major roles in the regulation of macrophage inflammatory gene expression. Chromatinmodifying enzymes have been associated with macrophage activation, and inflammation can be modulated by targeting these [100]. For instance, macrophage-specific deletion of HDAC3 leads to an M2-like pro-fibrotic phenotype, associated with increased plaque stability in a mouse atherosclerosis model [101]. Furthermore, the histone demethylase Jmjd3 erases the repressive H3K27 trimethylation mark in LPS-stimulated mouse macrophages, thus controlling cell differentiation and the inflammatory response [102]. IL-4 also stimulates Jmjd3 expression in mouse macrophages [103], and Jmjd3 appears to be essential for normal BMDM development and subsequent M2 polarization by regulating Irf4 trimethylation [104]. Jmjd3 is considered an important nexus in cellular aging, as it is upregulated in response to stress signals and links chromatin remodeling with induction of senescence marker p16Ink4a and inflammatory gene expression [105]. Aside from histone modifications, DNA methylation is key in regulating DNA accessibility as DNA hypomethylation generally confers accessibility to transcription factors. Aging is characterized by general DNA hypomethylation, although site-specific hypermethylation also occurs [106]. For example, aging appears to be accompanied by hypermethylation of autophagy gene promoters, leading to suppression of transcription [107]. Furthermore, atherosclerosis has been linked to mutations in DNA methylation-modifying enzymes, such as TET2 and DNMT3a, which affect macrophage function [108]. Mouse models indicate that homo- or heterozygous deletion of TET2 leads to increased atherosclerosis development and macrophages with a hyperinflammatory profile that directly contributes to disease [108,109]. Alterations in epigenetic status with advancing age are directly connected to processes of cellular metabolism [110]. Metabolites of common catabolic pathways supply several of the necessary cofactors that are used by epigenetic enzymes. For example, acetyl-CoA is a cofactor for histone acetyltransferases, and nuclear levels of acetyl-CoA associate with the acetylation state of histones and thus provide a mechanism of epigenetic regulation [111]. Additional intracellular metabolites, like S-adenosylmethionine (SAM), a-ketoglutarate, or NAD+, influence the activity of histoneand DNA-modifying enzymes and thereby potentially affect the regulation of epigenetic patterns in inflammatory cells [63]. Thus, metabolic alterations may also provide triggers that affect epigenetic patterns and thereby change the macrophage inflammatory phenotype and function with aging.

Clinically, rapamycin is used as an important inhibitor of T cell proliferation, but inhibition of mTOR activation essentially affects all metabolically active cells and mentioned side effects probably have multifaceted origins [73]. Restriction of essential amino acids, such as methionine or tryptophan, might be another way to improve macrophage function upon aging. Methionine and tryptophan restriction are both implicated in extending life span in mammals [68]. The tryptophan-degrading enzyme IDO expression is induced by IFN-g, but its expression may drive M2 polarization [74]. Methionine restriction seems to decrease macrophage migration and infiltration into WAT via increased expression of macrophage migration inhibition factor (MIF) [68]. Together, the effects of amino acid restriction on macrophage function appear to be complex and warrant further investigation. Dietary adjustments influence cellular function, but also mediate changes in microbiota composition. This can also be achieved by supplementation with pre- or probiotics. Bifidobacterium strain LKM512 promotes longevity in mice, mediated by upregulation of polyamine production by gut microbiota, leading to decreased tumor and ulcer incidence and gut permeability, possibly suppressing inflammaging via downregulation of the TNF/NF-kB pathway [75]. In accordance, a polyamine-rich diet was shown to promote longevity and inhibit age-associated pathologies such as glomerular atrophy in mice [76]. Polyamines are also needed for IL-4-induced expression of several M2 markers [77] and they stimulate autophagy [78]. Additionally, they inhibit the expression of pro-inflammatory factors in M1 macrophages. Microglia appear to be under constant control of gut microbiota, and defective microglia in mice lacking complex microbiota composition can be restored by microbiota and 10

Trends in Immunology, Month Year, Vol. xx, No. yy

TREIMM 1536 No. of Pages 15

Outstanding Questions

p62 SQSTM1

Resveratrol

AICAR

Meƞormin

Are macrophages the most important contributors to inflammatory cytokine production characterizing inflammaging?

Dietary restricƟon AMPK

Autophagy

mTOR

Rapamycin

SIRT1

Methionine tryptophan restricƟon

Polyamines ProbioƟcs

SCFA

Which are the dominant factors that drive the inflammaging process in macrophages? Are these intrinsic, related to macrophage aging, or extrinsic and derived from aging tissue? How is in vivo macrophage function affected in aging? Which steady state and induced macrophage functions are subject to alterations?

Pro-inflammatory cytokine producƟon

Figure 2. Putative Molecular Targets in Aging Macrophages to Modulate Inflammaging. Several treatments, applied as therapeutic, food additive, or altered nutrient intake (indicated in blue), have been shown to extend life span and decrease inflammation. These impact in particular on the nutrient sensor mammalian target of rapamycin (mTOR) (dietary restriction or methionine/tryptophan restriction), on the AMP-activated protein kinase (AMPK), or the NAD-dependent deacetylase sirtuin-1 (SIRT1) (metformin, AICAR, resveratrol) to inhibit production of pro-inflammatory cytokines and to stimulate autophagy. Resveratrol also stimulates p62/Sequestosome-1 (SQSTM1), and via this route, autophagy. Other molecular targets are involved in autophagy stimulation by polyamines, produced by probiotics or ingested with food. Another health-promoting contribution by probiotics is thought to be their production of short-chain fatty acids (SCFA), which inhibit pro-inflammatory cytokine production.

How do macrophage polarization states relate to the cells’ pro-inflammatory activity? And how does aging affect the ability of macrophages to be polarized in different directions? Could targeting aged macrophages, by pharmaceutical, nutraceutical, or pre- or probiotic ways, be used to reduce inflammaging?

microbiota-derived short chain fatty acids [79]. This indicates a close interaction between remote tissue macrophages and microbiota. Moreover, recent experiments involving transfer of microbiota from aged mice into young germ-free mice have shown that inflammaging is associated with increased levels of certain bacterial species such as TM7 and Proteobacteria in the aged gut microbiota, accompanied by increased TNF expression in the ileum, and is related to enhanced leakage of inflammatory bacterial components such as TLR2 ligands into circulation [80]. Together, these notions may favor studies examining the effect of probiotics, prebiotics, or fecal transplantation on aging animals, possibly in a macrophage-specific manner. Modulation of metabolism by therapeutic or dietary treatment might thus provide interesting approaches to increase longevity, albeit not necessarily providing clearly understood effects in macrophage profiles, particularly when studied in isolation.

Concluding Remarks We have aimed to address whether aging-related intrinsic changes in macrophages contribute significantly to the systemic condition of inflammaging (see Outstanding Questions). We posit that macrophages, as major initiators, effectors, and regulators of inflammatory responses, might be central players in this process [10,11]. This is supported by recent findings that depletion of macrophages from aged mice can alleviate tissue dysfunction and diminish increased cytokine production relative to young macrophages [7,8]. In tissues of aging mice an accumulation has been observed of macrophages with a remarkable M2-like, yet proinflammatory profile [27,28]. These cells express the senescence markers p16INK4a and Trends in Immunology, Month Year, Vol. xx, No. yy

11

TREIMM 1536 No. of Pages 15

b-galactosidase, but are considered non-senescent, since the phenotype is independent of p53 and can be reversed by triggering the cells. The degrees to which exogenous versus intrinsic changes contribute to the induction of the aged macrophage phenotype are difficult to define at present. We have reasoned that macrophages, especially long-lived tissue macrophages, do not escape intrinsic changes related to cellular damage at the genomic, proteomic, and lipidomic level. Bone marrow precursors from aged mice can be stimulated in vitro to proliferate extensively and give rise to mature macrophages, which show alterations in metabolism and responses to microbial ligands compared with BMDM generated from young adult mice. This indicates intrinsic differences are present at the precursor cell level and suggests similar changes likely occur in vivo. An experimental approach to shed light on this discussion regarding exogenous versus macrophage-intrinsic triggers might be to generate a mouse model in which fast aginginducing mutations, such as depletion of DNA repair enzyme Ercc1, are expressed in a macrophage-specific manner. Such a model might also provide insight into the question on whether the increase of pro-inflammatory mediators in the steady state that characterize inflammaging occurs primarily in senescent parenchymal cells or in macrophages. Macrophage depletion studies have already indicated that macrophages can contribute crucially to the age-associated systemic increase of inflammatory cytokines upon triggering [9]. In these experiments, aged mice showed increased cytokine production and succumbed to immune stimulation when compared with young mice that survived. This may highlight the elevated responsiveness of aged macrophages to the applied trigger, but does not address their contribution to the aged steady state. In view of the currently available experimental evidence, an attractive scenario describing the mechanism underlying alterations at the macrophage level during aging might involve the phenomenon of innate memory. This has been described recently as trained immunity, explaining enhanced responses, but also includes the longer known induction of tolerance [81,82]. Chronic exposure of long-lived tissue macrophages and bone marrow precursors to increasing levels of inflammatory triggers, such as LPS, S100A8/A9, and products from dying or senescent cells, might impose epigenetic changes that cause altered responsiveness of macrophages during aging. Together with intrinsic changes induced by a decreased ability to repair cellular damage, this might lead to an enhanced, or sometimes decreased, response to an acute trigger, such as that occurring from infection or metabolic perturbation. Acknowledgements This work is supported by grants from TI Food and Nutrition, a public–private partnership on precompetitive research in food and nutrition, ZonMW, the Netherlands Heart Foundation, and Spark-Holding BV, the European Union and Fondation Leducq. Size constraints, unfortunately, limited our possibilities to refer to many studies recently published in this rapidly expanding field of macrophage activation.

Supplemental Information Supplemental information associated with this article can be found online at https://doi.org/10.1016/j.it.2018.12.007.

References 1.

Lopez-Otin, C. et al. (2013) The hallmarks of aging. Cell 153, 1194–1217

2.

Cuervo, A.M. and Macian, F. (2014) Autophagy and the immune function in aging. Curr. Opin. Immunol. 29, 97–104

3.

Song, R. et al. (2018) The systems biology of single-cell aging. iScience 7, 154–169

12

Trends in Immunology, Month Year, Vol. xx, No. yy

4.

Franceschi, C. (2007) Inflammaging as a major characteristic of old people: can it be prevented or cured? Nutr. Rev. 65, S173– S176

5.

Coppe, J.P. et al. (2010) The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. 5, 99–118

TREIMM 1536 No. of Pages 15

29. Lavie, L. and Weinreb, O. (1996) Age- and strain-related changes in tissue transglutaminase activity in murine macrophages: the effects of inflammation and induction by retinol. Mech. Ageing Dev. 90, 129–143

6.

Biswas, S.K. and Mantovani, A. (2012) Orchestration of metabolism by macrophages. Cell Metab. 15, 432–437

7.

Woodcock, K.J. et al. (2015) Macrophage-derived upd3 cytokine causes impaired glucose homeostasis and reduced lifespan in Drosophila fed a lipid-rich diet. Immunity 42, 133–144

8.

Yuan, X. et al. (2018) Macrophage depletion ameliorates peripheral neuropathy in aging mice. J. Neurosci. 38, 4610–4620

30. Martinez, F.O. et al. (2013) Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences. Blood 121, e57–e69

9.

Bouchlaka, M.N. et al. (2013) Aging predisposes to acute inflammatory induced pathology after tumor immunotherapy. J. Exp. Med. 210, 2223–2237

31. Bains, W. (2013) Transglutaminse 2 and EGGL, the protein cross-link formed by transglutaminse 2, as therapeutic targets for disabilities of old age. Rejuvenation Res. 16, 495–517

10. Franceschi, C. et al. (2018) Inflammaging: a new immune-metabolic viewpoint for age-related diseases. Nat. Rev. Endocrinol. 14, 576–590

32. Man, A.L. et al. (2015) Age-associated modifications of intestinal permeability and innate immunity in human small intestine. Clin. Sci. (Lond.) 129, 515–527

11. Franceschi, C. et al. (2017) Inflammaging and ‘Garb-aging’. Trends Endocrinol. Metab. 28, 199–212

33. Rutkowski, K. et al. (2014) Dehydroepiandrosterone (DHEA): hypes and hopes. Drugs 74, 1195–1207

12. Kohut, M.L. et al. (2004) Age effects on macrophage function vary by tissue site, nature of stimulant, and exercise behavior. Exp. Gerontol. 39, 1347–1360

34. Rahat, M.A. et al. (2011) Molecular mechanisms regulating macrophage response to hypoxia. Front. Immunol. 2, 45

13. Lavin, Y. et al. (2014) Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell 159, 1312–1326 14. Lumeng, C.N. et al. (2011) Aging is associated with an increase in T cells and inflammatory macrophages in visceral adipose tissue. J. Immunol. 187, 6208–6216 15. Wang, Y. et al. (2015) Increases of M2a macrophages and fibrosis in aging muscle are influenced by bone marrow aging and negatively regulated by muscle-derived nitric oxide. Aging Cell 14, 678–688 16. Linehan, E. et al. (2014) Aging impairs peritoneal but not bone marrow-derived macrophage phagocytosis. Aging Cell 13, 699–708 17. Natrajan, M.S. et al. (2015) Retinoid X receptor activation reverses age-related deficiencies in myelin debris phagocytosis and remyelination. Brain 138, 3581–3597 18. Spadaro, O. et al. (2016) Growth hormone receptor deficiency protects against age-related NLRP3 inflammasome activation and immune senescence. Cell Rep. 14, 1571–1580 19. Pattabiraman, G. et al. (2017) Aging-associated dysregulation of homeostatic immune response termination (and not initiation). Aging Cell 16, 585–593 20. Boehmer, E.D. et al. (2005) Aging negatively skews macrophage TLR2- and TLR4-mediated pro-inflammatory responses without affecting the IL-2-stimulated pathway. Mech. Ageing Dev. 126, 1305–1313 21. Boyd, A.R. et al. (2012) Age-related defects in TLR2 signaling diminish the cytokine response by alveolar macrophages during murine pneumococcal pneumonia. Exp. Gerontol. 47, 507–518

35. Stranks, A.J. et al. (2015) Autophagy controls acquisition of aging features in macrophages. J. Innate Immun. 7, 375–391 36. Fei, F. et al. (2016) Age-associated metabolic dysregulation in bone marrow-derived macrophages stimulated with lipopolysaccharide. Sci. Rep. 6, 22637 37. Varol, C. et al. (2015) Macrophages: development and tissue specialization. Annu. Rev. Immunol. 33, 643–675 38. Naidoo, N. (2009) ER and aging-protein folding and the ER stress response. Ageing Res. Rev. 8, 150–159 39. Martinon, F. et al. (2010) TLR activation of the transcription factor XBP1 regulates innate immune responses in macrophages. Nat. Immunol. 11, 411–418 40. Shenderov, K. et al. (2014) Cutting edge: endoplasmic reticulum stress licenses macrophages to produce mature IL-1beta in response to TLR4 stimulation through a caspase-8- and TRIF-dependent pathway. J. Immunol. 192, 2029–2033 41. Fattah, E.A. et al. (2015) Critical role for IL-18 in spontaneous lung inflammation caused by autophagy deficiency. J. Immunol. 194, 5407–5416 42. Chang, C.P. et al. (2013) TLR2-dependent selective autophagy regulates NF-kappaB lysosomal degradation in hepatomaderived M2 macrophage differentiation. Cell Death Differ. 20, 515–523 43. Mahadevan, N.R. et al. (2011) Transmission of endoplasmic reticulum stress and pro-inflammation from tumor cells to myeloid cells. Proc. Natl. Acad. Sci. U. S. A. 108, 6561–6566 44. Xiu, F. et al. (2015) Prolonged endoplasmic reticulum-stressed hepatocytes drive an alternative macrophage polarization. Shock 44, 44–51

22. Sierra, A. et al. (2007) Microglia derived from aging mice exhibit an altered inflammatory profile. Glia 55, 412–424

45. Oh, J. et al. (2012) Endoplasmic reticulum stress controls M2 macrophage differentiation and foam cell formation. J. Biol. Chem. 287, 11629–11641

23. Chelvarajan, R.L. et al. (2006) Molecular basis of age-associated cytokine dysregulation in LPS-stimulated macrophages. J. Leukoc. Biol. 79, 1314–1327

46. Kapoor, N. et al. (2015) Transcription factors STAT6 and KLF4 implement macrophage polarization via the dual catalytic powers of MCPIP. J. Immunol. 194, 6011–6023

24. Van Beek, A.A. et al. (2016) Interaction of mouse splenocytes and macrophages with bacterial strains in vitro: the effect of age in the immune response. Benef. Microbes 7, 275–287

47. Chaudhari, N. et al. (2014) A molecular web: endoplasmic reticulum stress, inflammation, and oxidative stress. Front. Cell. Neurosci. 8, 213

25. Chen, H. et al. (2013) Elevated COX2 expression and PGE2 production by downregulation of RXRalpha in senescent macrophages. Biochem. Biophys. Res. Commun. 440, 157–162

48. Arruda, A.P. and Hotamisligil, G.S. (2015) Calcium homeostasis and organelle function in the pahorngthogenesis of obesity and diabetes. Cell Metab. 22, 381–397

26. Wu, D. et al. (2013) Diet-induced obesity has a differential effect on adipose tissue and macrophage inflammatory responses of young and old mice. Biofactors 39, 326–333

49. Horng, T. (2014) Calcium signaling and mitochondrial destabilization in the triggering of the NLRP3 inflammasome. Trends Immunol. 35, 253–261

27. Hall, B.M. et al. (2016) Aging of mice is associated with p16 (Ink4a)- and beta-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells. Aging (Albany N. Y.) 8, 1294–1315

50. Lavieri, R. et al. (2016) Redox stress unbalances the inflammatory cytokine network: role in autoinflammatory patients and healthy subjects. J. Leukoc. Biol. 99, 79–86

28. Hall, B.M. et al. (2017) p16(Ink4a) and senescence-associated beta-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli. Aging (Albany N. Y.) 9, 1867–1884

51. Loftus, R.M. and Finlay, D.K. (2016) Immunometabolism: cellular metabolism turns immune regulator. J. Biol. Chem. 291, 1–10 52. Pearce, E.L. and Pearce, E.J. (2013) Metabolic pathways in immune cell activation and quiescence. Immunity 38, 633–643

Trends in Immunology, Month Year, Vol. xx, No. yy

13

TREIMM 1536 No. of Pages 15

53. Van den Bossche, J. et al. (2017) Macrophage immunometabolism: where are we (going)? Trends Immunol. 38, 395–406 54. Mauer, J. et al. (2010) Myeloid cell-restricted insulin receptor deficiency protects against obesity-induced inflammation and systemic insulin resistance. PLoS Genet. 6, e1000938 55. Knuever, J. et al. (2015) Myeloid cell-restricted insulin/IGF-1 receptor deficiency protects against skin inflammation. J. Immunol. 195, 5296–5308 56. Youm, Y.H. et al. (2013) Canonical NLRP3 inflammasome links systemic low-grade inflammation to functional decline in aging. Cell Metab. 18, 519–532 57. Van den Bossche, J. et al. (2016) Mitochondrial dysfunction prevents repolarization of inflammatory macrophages. Cell Rep. 17, 684–696 58. O’Neill, L.A. and Hardie, D.G. (2013) Metabolism of inflammation limited by AMPK and pseudo-starvation. Nature 493, 346–355 59. Yeung, F. et al. (2004) Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase. EMBO J. 23, 2369–2380 60. Minhas, P.S. et al. (2018) Macrophage de novo NAD(+) synthesis specifies immune function in aging and inflammation. Nat. Immunol. 20, 50–63 61. Kelly, T.J. et al. (2009) GCN5-mediated transcriptional control of the metabolic coactivator PGC-1beta through lysine acetylation. J. Biol. Chem. 284, 19945–19952 62. Vats, D. et al. (2006) Oxidative metabolism and PGC-1beta attenuate macrophage-mediated inflammation. Cell Metab. 4, 13–24 63. Baardman, J. et al. (2015) Metabolic-epigenetic crosstalk in macrophage activation. Epigenomics 7, 1155–1164 64. Cheng, S.C. et al. (2014) mTOR- and HIF-1alpha-mediated aerobic glycolysis as metabolic basis for trained immunity. Science 345, 1250684 65. Mattison, J.A. et al. (2017) Caloric restriction improves health and survival of rhesus monkeys. Nat. Commun. 8, 14063 66. Jolly, C.A. (2004) Dietary restriction and immune function. J. Nutr. 134, 1853–1856 67. Starr, M.E. et al. (2016) Short-term dietary restriction rescues mice from lethal abdominal sepsis and endotoxemia and reduces the inflammatory/coagulant potential of adipose tissue. Crit. Care Med. 44, e509–e519 68. Wanders, D. et al. (2014) Transcriptional impact of dietary methionine restriction on systemic inflammation: relevance to biomarkers of metabolic disease during aging. Biofactors 40, 13–26 69. Yin, Z. et al. (2018) Low-fat diet with caloric restriction reduces white matter microglia activation during aging. Front. Mol. Neurosci. 11, 65 70. Zhang, Z. et al. (2010) Roles of SIRT1 in the acute and restorative phases following induction of inflammation. J. Biol. Chem. 285, 41391–41401 71. Wilkinson, J.E. et al. (2012) Rapamycin slows aging in mice. Aging Cell 11, 675–682 72. Mercalli, A. et al. (2013) Rapamycin unbalances the polarization of human macrophages to M1. Immunology 140, 179–190 73. Kaplan, B. et al. (2014) Strategies for the management of adverse events associated with mTOR inhibitors. Transplant. Rev. (Orlando) 28, 126–133 74. Wang, X.F. et al. (2014) The role of indoleamine 2,3-dioxygenase (IDO) in immune tolerance: focus on macrophage polarization of THP-1 cells. Cell. Immunol. 289, 42–48 75. Matsumoto, M. et al. (2011) Longevity in mice is promoted by probiotic-induced suppression of colonic senescence dependent on upregulation of gut bacterial polyamine production. PLoS One 6, e23652 76. Soda, K. et al. (2009) Polyamine-rich food decreases age-associated pathology and mortality in aged mice. Exp. Gerontol. 44, 727–732

14

Trends in Immunology, Month Year, Vol. xx, No. yy

77. Van den Bossche, J. et al. (2012) Pivotal advance: arginase-1independent polyamine production stimulates the expression of IL-4-induced alternatively activated macrophage markers while inhibiting LPS-induced expression of inflammatory genes. J. Leukoc. Biol. 91, 685–699 78. Pietrocola, F. et al. (2015) Spermidine induces autophagy by inhibiting the acetyltransferase EP300. Cell Death Differ. 22, 509–516 79. Erny, D. et al. (2015) Host microbiota constantly control maturation and function of microglia in the CNS. Nat. Neurosci. 18, 965–977 80. Fransen, F. et al. (2017) Aged gut microbiota contributes to systemical inflammaging after transfer to germ-free mice. Front. Immunol. 8, 1385 81. Franceschi, C. et al. (2017) Immunobiography and the heterogeneity of immune responses in the elderly: a focus on inflammaging and trained immunity. Front. Immunol. 8, 982 82. Bauer, M. et al. (2018) Remembering pathogen dose: long-term adaptation in innate immunity. Trends Immunol. 39, 438–445 83. Xue, J. et al. (2014) Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity 40, 274–288 84. Ginhoux, F. et al. (2016) New insights into the multidimensional concept of macrophage ontogeny, activation and function. Nat. Immunol. 17, 34–40 85. Vogelpoel, L.T. et al. (2014) Fc gamma receptor-TLR cross-talk elicits pro-inflammatory cytokine production by human M2 macrophages. Nat. Commun. 5, 5444 86. Hetz, C. (2012) The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat. Rev. Mol. Cell Biol. 13, 89–102 87. Hotamisligil, G.S. (2010) Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 140, 900–917 88. Kroemer, G. et al. (2010) Autophagy and the integrated stress response. Mol. Cell 40, 280–293 89. Kato, H. et al. (2012) mTORC1 serves ER stress-triggered apoptosis via selective activation of the IRE1-JNK pathway. Cell Death Differ. 19, 310–320 90. Ghosh, A.K. et al. (2015) Elevated endoplasmic reticulum stress response contributes to adipose tissue inflammation in aging. J. Gerontol. A Biol. Sci. Med. Sci. 70, 1320–1329 91. Freemerman, A.J. et al. (2014) Metabolic reprogramming of macrophages: glucose transporter 1 (GLUT1)-mediated glucose metabolism drives a proinflammatory phenotype. J. Biol. Chem. 289, 7884–7896 92. Tannahill, G.M. et al. (2013) Succinate is an inflammatory signal that induces IL-1beta through HIF-1alpha. Nature 496, 238–242 93. Huang, S.C. et al. (2016) Metabolic reprogramming mediated by the mTORC2-IRF4 signaling axis is essential for macrophage alternative activation. Immunity 45, 817–830 94. Tan, Z. et al. (2015) Pyruvate dehydrogenase kinase 1 participates in macrophage polarization via regulating glucose metabolism. J. Immunol. 194, 6082–6089 95. Covarrubias, A.J. et al. (2016) Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation. eLife 5, e11612 96. Namgaladze, D. and Brune, B. (2016) Macrophage fatty acid oxidation and its roles in macrophage polarization and fatty acidinduced inflammation. Biochim. Biophys. Acta 1861, 1796–1807 97. Nomura, M. et al. (2016) Fatty acid oxidation in macrophage polarization. Nat. Immunol. 17, 216–217 98. Fan, W. et al. (2010) FoxO1 regulates Tlr4 inflammatory pathway signalling in macrophages. EMBO J. 29, 4223–4236 99. Chung, S. et al. (2015) Distinct role of FoxO1 in M-CSF- and GM-CSF-differentiated macrophages contributes LPS-mediated IL-10: implication in hyperglycemia. J. Leukoc. Biol. 97, 327–339

TREIMM 1536 No. of Pages 15

100. Neele, A.E. et al. (2015) Epigenetic pathways in macrophages emerge as novel targets in atherosclerosis. Eur. J. Pharmacol. 763, 79–89 101. Hoeksema, M.A. et al. (2014) Targeting macrophage histone deacetylase 3 stabilizes atherosclerotic lesions. EMBO Mol. Med. 6, 1124–1132 102. De Santa, F. et al. (2009) Jmjd3 contributes to the control of gene expression in LPS-activated macrophages. EMBO J. 28, 3341–3352 103. Ishii, M. et al. (2009) Epigenetic regulation of the alternatively activated macrophage phenotype. Blood 114, 3244–3254 104. Satoh, T. et al. (2010) The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection. Nat. Immunol. 11, 936–944 105. Salminen, A. et al. (2014) Histone demethylase Jumonji D3 (JMJD3/KDM6B) at the nexus of epigenetic regulation of inflammation and the aging process. J. Mol. Med. (Berl.) 92, 1035–1043

106. Jung, M. and Pfeifer, G.P. (2015) Aging and DNA methylation. BMC Biol. 13, 7 107. Khalil, H. et al. (2016) Aging is associated with hypermethylation of autophagy genes in macrophages. Epigenetics 11, 381–388 108. Jaiswal, S. et al. (2017) Clonal hematopoiesis and risk of atherosclerotic cardiovascular disease. N. Engl. J. Med. 377, 111–121 109. Fuster, J.J. et al. (2017) Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice. Science 355, 842–847 110. Kaelin, W.G., Jr and McKnight, S.L. (2013) Influence of metabolism on epigenetics and disease. Cell 153, 56–69 111. Sutendra, G. et al. (2014) A nuclear pyruvate dehydrogenase complex is important for the generation of acetyl-CoA and histone acetylation. Cell 158, 84–97

Trends in Immunology, Month Year, Vol. xx, No. yy

15